High-Throughput Sequence Analysis of Turbot (Scophthalmus maximus) Transcriptome Using 454-Pyrosequencing for the Discovery of Antiviral Immune Genes

PubMed Central

Pereiro, Patricia; Balseiro, Pablo; Romero, Alejandro; Dios, Sonia; Forn-Cuni, Gabriel; Fuste, Berta; Planas, Josep V.; Beltran, Sergi; Novoa, Beatriz; Figueras, Antonio

2012-01-01

Background Turbot (Scophthalmus maximus L.) is an important aquacultural resource both in Europe and Asia. However, there is little information on gene sequences available in public databases. Currently, one of the main problems affecting the culture of this flatfish is mortality due to several pathogens, especially viral diseases which are not treatable. In order to identify new genes involved in immune defense, we conducted 454-pyrosequencing of the turbot transcriptome after different immune stimulations. Methodology/Principal Findings Turbot were injected with viral stimuli to increase the expression level of immune-related genes. High-throughput deep sequencing using 454-pyrosequencing technology yielded 915,256 high-quality reads. These sequences were assembled into 55,404 contigs that were subjected to annotation steps. Intriguingly, 55.16% of the deduced protein was not significantly similar to any sequences in the databases used for the annotation and only 0.85% of the BLASTx top-hits matched S. maximus protein sequences. This relatively low level of annotation is possibly due to the limited information for this specie and other flatfish in the database. These results suggest the identification of a large number of new genes in turbot and in fish in general. A more detailed analysis showed the presence of putative members of several innate and specific immune pathways. Conclusions/Significance To our knowledge, this study is the first transcriptome analysis using 454-pyrosequencing for turbot. Previously, there were only 12,471 EST and less of 1,500 nucleotide sequences for S. maximus in NCBI database. Our results provide a rich source of data (55,404 contigs and 181,845 singletons) for discovering and identifying new genes, which will serve as a basis for microarray construction, gene expression characterization and for identification of genetic markers to be used in several applications. Immune stimulation in turbot was very effective, obtaining an enormous variety of sequences belonging to genes involved in the defense mechanisms. PMID:22629298

Monitoring of microbial communities in anaerobic digestion sludge for biogas optimisation.

PubMed

Lim, Jun Wei; Ge, Tianshu; Tong, Yen Wah

2018-01-01

This study characterised and compared the microbial communities of anaerobic digestion (AD) sludge using three different methods - (1) Clone library; (2) Pyrosequencing; and (3) Terminal restriction fragment length polymorphism (T-RFLP). Although high-throughput sequencing techniques are becoming increasingly popular and affordable, the reliance of such techniques for frequent monitoring of microbial communities may be a financial burden for some. Furthermore, the depth of microbial analysis revealed by high-throughput sequencing may not be required for monitoring purposes. This study aims to develop a rapid, reliable and economical approach for the monitoring of microbial communities in AD sludge. A combined approach where genetic information of sequences from clone library was used to assign phylogeny to T-RFs determined experimentally was developed in this study. In order to assess the effectiveness of the combined approach, microbial communities determined by the combined approach was compared to that characterised by pyrosequencing. Results showed that both pyrosequencing and clone library methods determined the dominant bacteria phyla to be Proteobacteria, Firmicutes, Bacteroidetes, and Thermotogae. Both methods also found that sludge A and B were predominantly dominated by acetogenic methanogens followed by hydrogenotrophic methanogens. The number of OTUs detected by T-RFLP was significantly lesser than that detected by the clone library. In this study, T-RFLP analysis identified majority of the dominant species of the archaeal consortia. However, many of the more highly diverse bacteria consortia were missed. Nevertheless, the combined approach developed in this study where clone sequences from the clone library were used to assign phylogeny to T-RFs determined experimentally managed to accurately predict the same dominant microbial groups for both sludge A and sludge B, as compared to the pyrosequencing results. Results showed that the combined approach of clone library and T-RFLP accurately predicted the dominant microbial groups and thus is a reliable and more economical way to monitor the evolution of microbial systems in AD sludge. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tracing the evolution of degraders in activated sludge during the sludge’s acclimation to a xenobiotic organic

NASA Astrophysics Data System (ADS)

Chong, N. M.; Fan, C. H.; Yang, Y. C.

2017-01-01

The molecular biology method of high-throughput pyrosequencing was employed to examine the change of activated sludge community structures during the process in which activated sludge was acclimated to and degraded a target xenobiotic. The sample xenobiotic organic compound used as the activated sludge acclimation target was the herbicide 2,4-dichlorphenoxyacetic acid (2,4-D). Indigenous activated sludge microorganisms were acclimated to 2,4-D as the sole carbon source in both the batch and the continuous-flow reaction modes. Sludge masses at multiple time points during the course of acclimation were subjected to pyrosequencing targeting the microorganisms’ 16S rRNA genes. With the bacterial 16S rRNA sequencing results the genera that increased in abundance were checked with degradative pathway databases or literature to confirm that they are commonly seen as potent degraders of 2,4-D. From this systematic examination of degrader changes at time points during activated sludge acclimation and degradation of the target xenobiotic, the trend of degrader evolution in activated sludge over the sludge’s acclimation process to a xenobiotic was traced.

High-Throughput Amplicon Sequencing Reveals Distinct Communities within a Corroding Concrete Sewer System

PubMed Central

Dennis, Paul G.; Keller, Jurg; Tyson, Gene W.

2012-01-01

Microbially induced concrete corrosion (MICC) is an important problem in sewers. Here, small-subunit (SSU) rRNA gene amplicon pyrosequencing was used to characterize MICC communities. Microbial community composition differed between wall- and ceiling-associated MICC layers. Acidithiobacillus spp. were present at low abundances, and the communities were dominated by other sulfur-oxidizing-associated lineages. PMID:22843532

Transcriptomics of In Vitro Immune-Stimulated Hemocytes from the Manila Clam Ruditapes philippinarum Using High-Throughput Sequencing

PubMed Central

Moreira, Rebeca; Balseiro, Pablo; Planas, Josep V.; Fuste, Berta; Beltran, Sergi; Novoa, Beatriz; Figueras, Antonio

2012-01-01

Background The Manila clam (Ruditapes philippinarum) is a worldwide cultured bivalve species with important commercial value. Diseases affecting this species can result in large economic losses. Because knowledge of the molecular mechanisms of the immune response in bivalves, especially clams, is scarce and fragmentary, we sequenced RNA from immune-stimulated R. philippinarum hemocytes by 454-pyrosequencing to identify genes involved in their immune defense against infectious diseases. Methodology and Principal Findings High-throughput deep sequencing of R. philippinarum using 454 pyrosequencing technology yielded 974,976 high-quality reads with an average read length of 250 bp. The reads were assembled into 51,265 contigs and the 44.7% of the translated nucleotide sequences into protein were annotated successfully. The 35 most frequently found contigs included a large number of immune-related genes, and a more detailed analysis showed the presence of putative members of several immune pathways and processes like the apoptosis, the toll like signaling pathway and the complement cascade. We have found sequences from molecules never described in bivalves before, especially in the complement pathway where almost all the components are present. Conclusions This study represents the first transcriptome analysis using 454-pyrosequencing conducted on R. philippinarum focused on its immune system. Our results will provide a rich source of data to discover and identify new genes, which will serve as a basis for microarray construction and the study of gene expression as well as for the identification of genetic markers. The discovery of new immune sequences was very productive and resulted in a large variety of contigs that may play a role in the defense mechanisms of Ruditapes philippinarum. PMID:22536348

Rapid Molecular Identification of Human Taeniid Cestodes by Pyrosequencing Approach

PubMed Central

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

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

PubMed

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.

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

PubMed

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 < 0.05). The bacterial diversity in 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 < 0.05). The results showed that there was no one specific pathogen but rather pathogenic populations in plaque that significantly correlated with dental caries. The enormous diversity of 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.

Genome sequencing in microfabricated high-density picolitre reactors.

PubMed

Margulies, Marcel; Egholm, Michael; Altman, William E; Attiya, Said; Bader, Joel S; Bemben, Lisa A; Berka, Jan; Braverman, Michael S; Chen, Yi-Ju; Chen, Zhoutao; Dewell, Scott B; Du, Lei; Fierro, Joseph M; Gomes, Xavier V; Godwin, Brian C; He, Wen; Helgesen, Scott; Ho, Chun Heen; Ho, Chun He; Irzyk, Gerard P; Jando, Szilveszter C; Alenquer, Maria L I; Jarvie, Thomas P; Jirage, Kshama B; Kim, Jong-Bum; Knight, James R; Lanza, Janna R; Leamon, John H; Lefkowitz, Steven M; Lei, Ming; Li, Jing; Lohman, Kenton L; Lu, Hong; Makhijani, Vinod B; McDade, Keith E; McKenna, Michael P; Myers, Eugene W; Nickerson, Elizabeth; Nobile, John R; Plant, Ramona; Puc, Bernard P; Ronan, Michael T; Roth, George T; Sarkis, Gary J; Simons, Jan Fredrik; Simpson, John W; Srinivasan, Maithreyan; Tartaro, Karrie R; Tomasz, Alexander; Vogt, Kari A; Volkmer, Greg A; Wang, Shally H; Wang, Yong; Weiner, Michael P; Yu, Pengguang; Begley, Richard F; Rothberg, Jonathan M

2005-09-15

The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.

Pyrosequencing-based validation of a simple cell-suspension polymerase chain reaction assay for Campylobacter with application of high-processivity polymerase and novel internal amplification controls for rapid and specific detection.

PubMed

Oakley, Brian B; Line, J Eric; Berrang, Mark E; Johnson, Jessica M; Buhr, R Jeff; Cox, Nelson A; Hiett, Kelli L; Seal, Bruce S

2012-02-01

Although Campylobacter is an important food-borne human pathogen, there remains a lack of molecular diagnostic assays that are simple to use, cost-effective, and provide rapid results in research, clinical, or regulatory laboratories. Of the numerous Campylobacter assays that do exist, to our knowledge none has been empirically tested for specificity using high-throughput sequencing. Here we demonstrate the power of next-generation sequencing to determine the specificity of a widely cited Campylobacter-specific polymerase chain reaction (PCR) assay and describe a rapid method for direct cell suspension PCR to quickly and easily screen samples for Campylobacter. We present a specific protocol which eliminates the need for time-consuming and expensive genomic DNA extractions and, using a high-processivity polymerase, demonstrate conclusive screening of samples in <1 h. Pyrosequencing results show the assay to be extremely (>99%) sensitive, and spike-back experiments demonstrated a detection threshold of <10(2) CFU mL(-1). Additionally, we present 2 newly designed broad-range bacterial primer sets targeting the 23S rRNA gene that have wide applicability as internal amplification controls. Empirical testing of putative taxon-specific assays using high-throughput sequencing is an important validation step that is now financially feasible for research, regulatory, or clinical applications. Published by Elsevier Inc.

Population and community structure shifts of ammonia oxidizers after four-year successive biochar application to agricultural acidic and alkaline soils.

PubMed

He, Lili; Bi, Yucui; Zhao, Jin; Pittelkow, Cameron M; Zhao, Xu; Wang, Shenqiang; Xing, Guangxi

2018-04-01

Long-term studies that advance our mechanistic understanding of biochar (BC)‑nitrogen (N) interactions in agricultural soils are lacking. In this study, soil potential nitrification rates (PNR), the abundance and composition of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) communities following 4-year of BC application were investigated using the shaken-slurry procedure and molecular sequencing techniques for an acidic Oxisol (QU) and an alkaline Cambisol (YU). Soils were obtained from an outdoor soil column experiment with straw-BC application rates of 0 (BC0), 2.25 (BC2.25) and 11.3 (BC11.3) Mgha -1 per cropping season for eight consecutive wheat/millet seasons. Quantitative polymerase chain reaction (qPCR) and 454 high-throughput pyrosequencing techniques were performed to quantify and sequence amoA gene copies and composition of AOA and AOB. Results showed that QU had lower PNR and a higher ratio of amoA gene copies of AOA to AOB than YU, PNR of QU with BC application was significantly associated with the amoA gene of AOB. Similar to previous short-term findings, BC application enhanced QU soil nitrification, which may be explained by the significant increase in AOB abundance and a shift in AOB community structure from Nitrosospira cluster 2 toward cluster 3, along with the disappearance of some obligate acidophile AOA groups, leading to the appearance of ammonia-oxidizers from neutral-alkaline soils in BC-amended acid soils. Canonical correspondence analysis (CCA) showed that soil pH was the most important factor driving shifts in ammonia-oxidizers composition. Although BC application did not have significant effects on PNR in YU, BC11.3 decreased AOA and AOB gene copies and influenced the relative abundance of community structure. Our findings represent the first investigation of long-term BC effects on AOA and AOB communities in agricultural soils using 454 high-throughput pyrosequencing, showing that BC application can alter soil characteristics and influence ammonia oxidizer community composition, abundance, especially in acid soils. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of Microbial Communities in Open and Closed Circuit Bioelectrochemical MBRs by High-Throughput 454 Pyrosequencing

PubMed Central

Huang, Jian; Wang, Zhiwei; Zhu, Chaowei; Ma, Jinxing; Zhang, Xingran; Wu, Zhichao

2014-01-01

Two bioelectrochemical membrane bioreactors (MBRs) developed by integrating microbial fuel cell and MBR technology were operated under closed-circuit and open-circuit modes, and high-throughput 454 pyrosequencing was used to investigate the effects of the power generation on the microbial community of bio-anode and bio-cathode. Microbes on the anode under open-circuit operation (AO) were enriched and highly diverse when compared to those on the anode under closed-circuit operation (AC). However, among the cathodes the closed-circuit mode (CC) had richer and more diverse microbial community compared to the cathode under open-circuit mode (CO). On the anodes AO and AC, Proteobacteria and Bacteroidetes were the dominant phyla, while Firmicutes was enriched only on AC. Deltaproteobacteria affiliated to Proteobacteria were also more abundant on AC than AO. Furthermore, the relative abundance of Desulfuromonas, which are well-known electrogenic bacteria, were much higher on AC (10.2%) when compared to AO (0.11%), indicating that closed-circuit operation was more conducive for the growth of electrogenic bacteria on the anodes. On the cathodes, Protebacteria was robust on CC while Bacteroidetes was more abundant on CO. Rhodobacter and Hydrogenophaga were also enriched on CC than CO, suggesting that these genera play a role in electron transfer from the cathode surface to the terminal electron acceptors in the bioelectrochemical MBR under closed-circuit operation. PMID:24705450

Bacterial Community Profiling of H2/CO2 or Formate-Utilizing Acetogens Enriched from Diverse Ecosystems

NASA Astrophysics Data System (ADS)

Han, R.; Zhang, L.; Fu, B.; Liu, H.

2014-12-01

Synthetic gases are usually generated from either cellulosic agricultural waste combustion or industrial release and could be subsequently transformed into acetate, ethanol, and/or butyrate by homoacetogenic bacteria, which commonly possess reductive acetyl-CoA synthesis pathway. Homoacetogen-based syngas fermentation technology provides an alternative solution to link greenhouse gas emission control and cellulosic solid waste treatment with biofuels production. The objective of our current project is to hunt for homoacetogens with capabilities of highly efficiently converting syngases to chemical solvents. In this study, we evaluated homoacetogens population dynamics during enrichments and pinpointed dominant homoacetogens representing diverse ecosystems enriched by different substrates. We enriched homoacetogens from four different samples including waste activate sludge, freshwater sediment, anaerobic methanogenic sludge, and cow manure using H2/CO2 (4:1) or formate as substrate for homoacetogen enrichment. Along with the formyltetrahydrofolate synthetase (FTHFS) gene (fhs gene)-specific real time qPCR assay and Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis, 16S rRNA based 454 high-throughput pyrosequencing was applied to reveal the population dynamic and community structure during enrichment from different origins. Enrichment of homoacetogenic populations coincided with accumulations of short chain fatty acids such as acetate and butyrate. 454 high-throughput pyrosequencing revealed Firmicutes and Spirochaetes populations became dominant while the overall microbial diversity decreased after enrichment. The most abundant sequences among the four origins belonged to the following phyla: Firmicutes, Spirochaetes, Proteobacteria, and Bacteroidetes, accounting for 62.1%-99.1% of the total reads. The major putative homoacetogenic species enriched on H2/CO2 or formate belonged to Clostridium spp., Acetobacterium spp., Acetoanaerobium spp., Eubacterium spp., Sporomusa spp. This comprehensive molecular ecology study on homoacetogen enrichments provides molecular evidences for shaping homoacetogenic populations and targeting novel homoacetogenic species enriched from diverse ecosystems.

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

PubMed

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

2012-06-01

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

Rapid Genome Analyses of Emergent Human Adenovirus 14a Causing 2006-7 Febrile Respiratory Illness (FRI) Outbreaks in the US via High Throughput Next-Generation Pyrosequencing Technique

DTIC Science & Technology

2008-12-01

David Metzgar2), Kevin L. Russell2), Adriana Kajon3), Kuei-Hsiang Lin4) and Julia A. Lynch1). Division of Viral Diseases, Walter Reed Army...Puc BP, Ronan MT, Roth GT, Sarkis GJ, Simons JF, Simpson JW, Srinivasan M, Tartaro KR, Tomasz A, Vogt KA , Volkmer GA, Wang SH, Weiner MP, Yu P

Pyrosequencing of mcrA and Archaeal 16S rRNA Genes Reveals Diversity and Substrate Preferences of Methanogen Communities in Anaerobic Digesters

PubMed Central

Wilkins, David; Lu, Xiao-Ying; Shen, Zhiyong; Chen, Jiapeng

2014-01-01

Methanogenic archaea play a key role in biogas-producing anaerobic digestion and yet remain poorly taxonomically characterized. This is in part due to the limitations of low-throughput Sanger sequencing of a single (16S rRNA) gene, which in the past may have undersampled methanogen diversity. In this study, archaeal communities from three sludge digesters in Hong Kong and one wastewater digester in China were examined using high-throughput pyrosequencing of the methyl coenzyme M reductase (mcrA) and 16S rRNA genes. Methanobacteriales, Methanomicrobiales, and Methanosarcinales were detected in each digester, indicating that both hydrogenotrophic and acetoclastic methanogenesis was occurring. Two sludge digesters had similar community structures, likely due to their similar design and feedstock. Taxonomic classification of the mcrA genes suggested that these digesters were dominated by acetoclastic methanogens, particularly Methanosarcinales, while the other digesters were dominated by hydrogenotrophic Methanomicrobiales. The proposed euryarchaeotal order Methanomassiliicoccales and the uncultured WSA2 group were detected with the 16S rRNA gene, and potential mcrA genes for these groups were identified. 16S rRNA gene sequencing also recovered several crenarchaeotal groups potentially involved in the initial anaerobic digestion processes. Overall, the two genes produced different taxonomic profiles for the digesters, while greater methanogen richness was detected using the mcrA gene, supporting the use of this functional gene as a complement to the 16S rRNA gene to better assess methanogen diversity. A significant positive correlation was detected between methane production and the abundance of mcrA transcripts in digesters treating sludge and wastewater samples, supporting the mcrA gene as a biomarker for methane yield. PMID:25381241

High throughput pyrosequencing technology for molecular differential detection of Babesia vogeli, Hepatozoon canis, Ehrlichia canis and Anaplasma platys in canine blood samples.

PubMed

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.

SeqTrim: a high-throughput pipeline for pre-processing any type of sequence read

PubMed Central

2010-01-01

Background High-throughput automated sequencing has enabled an exponential growth rate of sequencing data. This requires increasing sequence quality and reliability in order to avoid database contamination with artefactual sequences. The arrival of pyrosequencing enhances this problem and necessitates customisable pre-processing algorithms. Results SeqTrim has been implemented both as a Web and as a standalone command line application. Already-published and newly-designed algorithms have been included to identify sequence inserts, to remove low quality, vector, adaptor, low complexity and contaminant sequences, and to detect chimeric reads. The availability of several input and output formats allows its inclusion in sequence processing workflows. Due to its specific algorithms, SeqTrim outperforms other pre-processors implemented as Web services or standalone applications. It performs equally well with sequences from EST libraries, SSH libraries, genomic DNA libraries and pyrosequencing reads and does not lead to over-trimming. Conclusions SeqTrim is an efficient pipeline designed for pre-processing of any type of sequence read, including next-generation sequencing. It is easily configurable and provides a friendly interface that allows users to know what happened with sequences at every pre-processing stage, and to verify pre-processing of an individual sequence if desired. The recommended pipeline reveals more information about each sequence than previously described pre-processors and can discard more sequencing or experimental artefacts. PMID:20089148

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

PubMed

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.

Deep-branching Novel Lineages and High Diversity of Haptophytes in the Skagerrak (Norway) Uncovered by 454 Pyrosequencing

PubMed Central

Egge, Elianne S; Eikrem, Wenche; Edvardsen, Bente

2015-01-01

Microalgae in the division Haptophyta may be difficult to identify to species by microscopy because they are small and fragile. Here, we used high-throughput sequencing to explore the diversity of haptophytes in outer Oslofjorden, Skagerrak, and supplemented this with electron microscopy. Nano- and picoplanktonic subsurface samples were collected monthly for 2 yr, and the haptophytes were targeted by amplification of RNA/cDNA with Haptophyta-specific 18S ribosomal DNA V4 primers. Pyrosequencing revealed higher species richness of haptophytes than previously observed in the Skagerrak by microscopy. From ca. 400,000 reads we obtained 156 haptophyte operational taxonomic units (OTUs) after rigorous filtering and 99.5% clustering. The majority (84%) of the OTUs matched environmental sequences not linked to a morphological species, most of which were affiliated with the order Prymnesiales. Phylogenetic analyses including Oslofjorden OTUs and available cultured and environmental haptophyte sequences showed that several of the OTUs matched sequences forming deep-branching lineages, potentially representing novel haptophyte classes. Pyrosequencing also retrieved cultured species not previously reported by microscopy in the Skagerrak. Electron microscopy revealed species not yet genetically characterised and some potentially novel taxa. This study contributes to linking genotype to phenotype within this ubiquitous and ecologically important protist group, and reveals great, unknown diversity. PMID:25099994

Effect of flow rate on growth and oxygen consumption of biofilm in gravity sewer.

PubMed

Xu, Jingwei; Li, Muzhi; He, Qiang; Sun, Xingfu; Zhou, Xiangren; Su, Zhenping; Ai, Hainan

2017-01-01

The function of sewer as reactors must rely on the biofilm in it. In this paper, the formation, structure, oxygen transfer, and activity of the biofilm under different hydraulic conditions were studied by the microelectrode technology, oxygen uptake rate (OUR) technology, and 454 high-throughput pyrosequencing technology. Results showed that when the wall-shear stresses were 1.12, 1.29, and 1.45 Pa, the porosity of the steady-state biofilm were 69.1, 64.4, and 55.1 %, respectively. The maximum values of OUR were 0.033, 0.027, and 0.022 mg/(L*s), respectively, and the COD removal efficiency in the sewers reached 40, 35, and 32 %, respectively. The research findings had an important significance on how to improve the treatment efficiency of the sewers. Fig. a Graphical Abstract.

Nitrogen amendments have predictable effects on soil microbial communities and processes

NASA Astrophysics Data System (ADS)

Ramirez, K. S.; Craine, J. M.; Fierer, N.

2011-12-01

Ecosystems worldwide are receiving increasing amounts of reactive nitrogen (N) through anthropogenic activities. While there has been much effort devoted to quantifying aboveground impacts of anthropogenic N effects, less work has focused on identifying belowground impacts. Bacteria play critical roles in ecosystem processes and identifying how anthropogenic N impacts bacterial communities may elucidate how critical microbially-mediated ecosystem functions are altered by N additions. In order to connect changes in soil processes to changes in the microbial community, we need to first determine if the changes are consistent across different soil types and ecosystems. We assessed the patterns of N effects across a variety of ecosystems in two ways. First, utilizing long-term experimental N gradients at Cedar Creek LTER, MN and Kellogg Biological Station LTER, MI, we examined the response of microbial communities to anthropogenic N additions. Using high-throughput pyrosequencing techniques we quantified changes in soil microbial communities across the nitrogen gradients. We observed strong directional shifts in community composition at both sites; N fertilization consistently impacted both the phylogenetic and taxonomic structure of soil bacterial community structure in a predictable manner regardless of ecosystem type. For example, at both sites Acidobacteria experienced significant declines as nitrogen increased, while other groups such as Actinobacteria and Bacteroidetes increased in relative abundance. Our results suggest that bacterial communities across these N fertility gradients are structured by either nitrogen and/or soil carbon availability, rather than by shifts in the plant community or soil pH indirectly associated with the elevated nitrogen inputs. Still, this field-work does not incorporate changes in soil processes (e.g. soil respiration) or microbial activity (e.g. microbial biomass and extracellular enzyme activity), or separate N from C effects. To address these factors, we performed a lab experiment, amending 30 soils collected from across North America with inorganic N. From this year-long incubation we obtained soil respiration, microbial biomass, bacterial community and extracellular enzyme activity measurements. Across all soil types we consistently observed a significant decrease in both soil respiration, approximately 10%, and microbial biomass, approximately 35%. Using high-throughput pyrosequencing we identified seven bacterial groups that responded significantly to the N additions, including those observed in our field survey. Together, this work suggests that increases in soil N shifts the functional capabilities of the microbial community and highlights possibly mechanisms behind the observed changes.

High-Throughput Sequencing of 16S rRNA Gene Amplicons: Effects of Extraction Procedure, Primer Length and Annealing Temperature

PubMed Central

Sergeant, Martin J.; Constantinidou, Chrystala; Cogan, Tristan; Penn, Charles W.; Pallen, Mark J.

2012-01-01

The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers. PMID:22666455

High-throughput sequencing of 16S rRNA gene amplicons: effects of extraction procedure, primer length and annealing temperature.

PubMed

Sergeant, Martin J; Constantinidou, Chrystala; Cogan, Tristan; Penn, Charles W; Pallen, Mark J

2012-01-01

The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers.

Pyrosequencing for detection of lamivudine-resistant hepatitis B virus.

PubMed

Lindström, Anna; Odeberg, Jacob; Albert, Jan

2004-10-01

Chronic hepatitis B virus (HBV) infection can cause severe liver disease, including cirrhosis and hepatocellular carcinoma. Lamivudine is a relatively recent alternative to alpha interferon for the treatment of HBV infection, but unfortunately, resistance to lamivudine commonly develops during monotherapy. Lamivudine-resistant HBV mutants display specific mutations in the YMDD (tyrosine, methionine, aspartate, aspartate) motif of the viral polymerase (reverse transcriptase [rt]), which is the catalytic site of the enzyme, i.e., methionine 204 to isoleucine (rtM204I) or valine (rtM204V). The latter mutation is often accompanied by a compensatory leucine-to-methionine change at codon 180 (rtL180M). In the present study, a novel sequencing method, pyrosequencing, was applied to the detection of lamivudine resistance mutations and was compared with direct Sanger sequencing. The new pyrosequencing method had advantages in terms of throughput. Experiments with mixtures of wild-type and resistant viruses indicated that pyrosequencing can detect minor sequence variants in heterogeneous virus populations. The new pyrosequencing method was evaluated with a small number of patient samples, and the results showed that the method could be a useful tool for the detection of lamivudine resistance in the clinical setting.

Dominant Tree Species and Soil Type Affect the Fungal Community Structure in a Boreal Peatland Forest.

PubMed

Sun, Hui; Terhonen, Eeva; Kovalchuk, Andriy; Tuovila, Hanna; Chen, Hongxin; Oghenekaro, Abbot O; Heinonsalo, Jussi; Kohler, Annegret; Kasanen, Risto; Vasander, Harri; Asiegbu, Fred O

2016-05-01

Boreal peatlands play a crucial role in global carbon cycling, acting as an important carbon reservoir. However, little information is available on how peatland microbial communities are influenced by natural variability or human-induced disturbances. In this study, we have investigated the fungal diversity and community structure of both the organic soil layer and buried wood in boreal forest soils using high-throughput sequencing of the internal transcribed spacer (ITS) region. We have also compared the fungal communities during the primary colonization of wood with those of the surrounding soils. A permutational multivariate analysis of variance (PERMANOVA) confirmed that the community composition significantly differed between soil types (P< 0.001) and tree species (P< 0.001). The distance-based linear models analysis showed that environmental variables were significantly correlated with community structure (P< 0.04). The availability of soil nutrients (Ca [P= 0.002], Fe [P= 0.003], and P [P= 0.003]) within the site was an important factor in the fungal community composition. The species richness in wood was significantly lower than in the corresponding soil (P< 0.004). The results of the molecular identification were supplemented by fruiting body surveys. Seven of the genera of Agaricomycotina identified in our surveys were among the top 20 genera observed in pyrosequencing data. Our study is the first, to our knowledge, fungal high-throughput next-generation sequencing study performed on peatlands; it further provides a baseline for the investigation of the dynamics of the fungal community in the boreal peatlands. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Dominant Tree Species and Soil Type Affect the Fungal Community Structure in a Boreal Peatland Forest

PubMed Central

Terhonen, Eeva; Kovalchuk, Andriy; Tuovila, Hanna; Chen, Hongxin; Oghenekaro, Abbot O.; Heinonsalo, Jussi; Kohler, Annegret; Kasanen, Risto; Vasander, Harri; Asiegbu, Fred O.

2016-01-01

Boreal peatlands play a crucial role in global carbon cycling, acting as an important carbon reservoir. However, little information is available on how peatland microbial communities are influenced by natural variability or human-induced disturbances. In this study, we have investigated the fungal diversity and community structure of both the organic soil layer and buried wood in boreal forest soils using high-throughput sequencing of the internal transcribed spacer (ITS) region. We have also compared the fungal communities during the primary colonization of wood with those of the surrounding soils. A permutational multivariate analysis of variance (PERMANOVA) confirmed that the community composition significantly differed between soil types (P < 0.001) and tree species (P < 0.001). The distance-based linear models analysis showed that environmental variables were significantly correlated with community structure (P < 0.04). The availability of soil nutrients (Ca [P = 0.002], Fe [P = 0.003], and P [P = 0.003]) within the site was an important factor in the fungal community composition. The species richness in wood was significantly lower than in the corresponding soil (P < 0.004). The results of the molecular identification were supplemented by fruiting body surveys. Seven of the genera of Agaricomycotina identified in our surveys were among the top 20 genera observed in pyrosequencing data. Our study is the first, to our knowledge, fungal high-throughput next-generation sequencing study performed on peatlands; it further provides a baseline for the investigation of the dynamics of the fungal community in the boreal peatlands. PMID:26896139

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

PubMed

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.

Pyrosequencing the Canine Faecal Microbiota: Breadth and Depth of Biodiversity

PubMed Central

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

Analysis of the melon (Cucumis melo) small RNAome by high-throughput pyrosequencing

PubMed Central

2011-01-01

Background Melon (Cucumis melo L.) is a commercially important fruit crop that is cultivated worldwide. The melon research community has recently benefited from the determination of a complete draft genome sequence and the development of associated genomic tools, which have allowed us to focus on small RNAs (sRNAs). These are short, non-coding RNAs 21-24 nucleotides in length with diverse physiological roles. In plants, they regulate gene expression and heterochromatin assembly, and control protection against virus infection. Much remains to be learned about the role of sRNAs in melon. Results We constructed 10 sRNA libraries from two stages of developing ovaries, fruits and photosynthetic cotyledons infected with viruses, and carried out high-throughput pyrosequencing. We catalogued and analysed the melon sRNAs, resulting in the identification of 26 known miRNA families (many conserved with other species), the prediction of 84 melon-specific miRNA candidates, the identification of trans-acting siRNAs, and the identification of chloroplast, mitochondrion and transposon-derived sRNAs. In silico analysis revealed more than 400 potential targets for the conserved and novel miRNAs. Conclusion We have discovered and analysed a large number of conserved and melon-specific sRNAs, including miRNAs and their potential target genes. This provides insight into the composition and function of the melon small RNAome, and paves the way towards an understanding of sRNA-mediated processes that regulate melon fruit development and melon-virus interactions. PMID:21812964

Proteolytic bacterial dominance in a full-scale municipal solid waste anaerobic reactor assessed by 454 pyrosequencing technology.

PubMed

Cardinali-Rezende, Juliana; Rojas-Ojeda, Patricia; Nascimento, Andréa M A; Sanz, José L

2016-03-01

Biomethanization entails a good means to reduce the organic fraction (OF) derived from municipal solid wastes (MSW). The bacterial diversity of a full scale MSW anaerobic reactor located in Madrid (Spain) was investigated using high-throughput 454 pyrosequencing. Even though the proteolytic bacteria prevailed throughout all of the process, community shifts were observed from the start-up to the steady-state conditions, with an increasing biodiversity displayed over time. The Bacteroidetes and the Firmicutes were the majority phyla: 55.1 and 40.2% (start-up) and 18.7 and 78.7 (steady-state) of the total reads. The system's lack of evenness remains noteworthy as the sequences affiliated to the proteolytic non-saccharolytic Proteiniphylum, Gallicola and Fastidiosipila genera, together with the saccharolytic Saccharofermentans, were predominant on the system and this predominance appears to correlate with the presence of a high ammonium concentration. The 454 pyrosequencing revealed a great diversity of rare organisms which seemingly do not sustain any metabolic roles in the course of the OF-MSW degradation. However, this scarce and unique microbiota can confer great resilience to the system as a buffer against nutritional and environmental changing conditions, thus opening the door to increase the current knowledge about the bacterial community dynamics taking place during MSW treatment processes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Random Amplification and Pyrosequencing for Identification of Novel Viral Genome Sequences

PubMed Central

Hang, Jun; Forshey, Brett M.; Kochel, Tadeusz J.; Li, Tao; Solórzano, Víctor Fiestas; Halsey, Eric S.; Kuschner, Robert A.

2012-01-01

ssRNA viruses have high levels of genomic divergence, which can lead to difficulty in genomic characterization of new viruses using traditional PCR amplification and sequencing methods. In this study, random reverse transcription, anchored random PCR amplification, and high-throughput pyrosequencing were used to identify orthobunyavirus sequences from total RNA extracted from viral cultures of acute febrile illness specimens. Draft genome sequence for the orthobunyavirus L segment was assembled and sequentially extended using de novo assembly contigs from pyrosequencing reads and orthobunyavirus sequences in GenBank as guidance. Accuracy and continuous coverage were achieved by mapping all reads to the L segment draft sequence. Subsequently, RT-PCR and Sanger sequencing were used to complete the genome sequence. The complete L segment was found to be 6936 bases in length, encoding a 2248-aa putative RNA polymerase. The identified L segment was distinct from previously published South American orthobunyaviruses, sharing 63% and 54% identity at the nucleotide and amino acid level, respectively, with the complete Oropouche virus L segment and 73% and 81% identity at the nucleotide and amino acid level, respectively, with a partial Caraparu virus L segment. The result demonstrated the effectiveness of a sequence-independent amplification and next-generation sequencing approach for obtaining complete viral genomes from total nucleic acid extracts and its use in pathogen discovery. PMID:22468136

Distinct soil microbial diversity under long-term organic and conventional farming

PubMed Central

Hartmann, Martin; Frey, Beat; Mayer, Jochen; Mäder, Paul; Widmer, Franco

2015-01-01

Low-input agricultural systems aim at reducing the use of synthetic fertilizers and pesticides in order to improve sustainable production and ecosystem health. Despite the integral role of the soil microbiome in agricultural production, we still have a limited understanding of the complex response of microbial diversity to organic and conventional farming. Here we report on the structural response of the soil microbiome to more than two decades of different agricultural management in a long-term field experiment using a high-throughput pyrosequencing approach of bacterial and fungal ribosomal markers. Organic farming increased richness, decreased evenness, reduced dispersion and shifted the structure of the soil microbiota when compared with conventionally managed soils under exclusively mineral fertilization. This effect was largely attributed to the use and quality of organic fertilizers, as differences became smaller when conventionally managed soils under an integrated fertilization scheme were examined. The impact of the plant protection regime, characterized by moderate and targeted application of pesticides, was of subordinate importance. Systems not receiving manure harboured a dispersed and functionally versatile community characterized by presumably oligotrophic organisms adapted to nutrient-limited environments. Systems receiving organic fertilizer were characterized by specific microbial guilds known to be involved in degradation of complex organic compounds such as manure and compost. The throughput and resolution of the sequencing approach permitted to detect specific structural shifts at the level of individual microbial taxa that harbours a novel potential for managing the soil environment by means of promoting beneficial and suppressing detrimental organisms. PMID:25350160

Stratified Microbial Structure and Activity in Sulfide- and Methane-Producing Anaerobic Sewer Biofilms

PubMed Central

Sun, Jing; Hu, Shihu; Sharma, Keshab Raj; Ni, Bing-Jie

2014-01-01

Simultaneous production of sulfide and methane by anaerobic sewer biofilms has recently been observed, suggesting that sulfate-reducing bacteria (SRB) and methanogenic archaea (MA), microorganisms known to compete for the same substrates, can coexist in this environment. This study investigated the community structures and activities of SRB and MA in anaerobic sewer biofilms (average thickness of 800 μm) using a combination of microelectrode measurements, molecular techniques, and mathematical modeling. It was seen that sulfide was mainly produced in the outer layer of the biofilm, between the depths of 0 and 300 μm, which is in good agreement with the distribution of SRB population as revealed by cryosection-fluorescence in situ hybridization (FISH). SRB had a higher relative abundance of 20% on the surface layer, which decreased gradually to below 3% at a depth of 400 μm. In contrast, MA mainly inhabited the inner layer of the biofilm. Their relative abundances increased from 10% to 75% at depths of 200 μm and 700 μm, respectively, from the biofilm surface layer. High-throughput pyrosequencing of 16S rRNA amplicons showed that SRB in the biofilm were mainly affiliated with five genera, Desulfobulbus, Desulfomicrobium, Desulfovibrio, Desulfatiferula, and Desulforegula, while about 90% of the MA population belonged to the genus Methanosaeta. The spatial organizations of SRB and MA revealed by pyrosequencing were consistent with the FISH results. A biofilm model was constructed to simulate the SRB and MA distributions in the anaerobic sewer biofilm. The good fit between model predictions and the experimental data indicate that the coexistence and spatial structure of SRB and MA in the biofilm resulted from the microbial types and their metabolic transformations and interactions with substrates. PMID:25192994

Stratified microbial structure and activity in sulfide- and methane-producing anaerobic sewer biofilms.

PubMed

Sun, Jing; Hu, Shihu; Sharma, Keshab Raj; Ni, Bing-Jie; Yuan, Zhiguo

2014-11-01

Simultaneous production of sulfide and methane by anaerobic sewer biofilms has recently been observed, suggesting that sulfate-reducing bacteria (SRB) and methanogenic archaea (MA), microorganisms known to compete for the same substrates, can coexist in this environment. This study investigated the community structures and activities of SRB and MA in anaerobic sewer biofilms (average thickness of 800 μm) using a combination of microelectrode measurements, molecular techniques, and mathematical modeling. It was seen that sulfide was mainly produced in the outer layer of the biofilm, between the depths of 0 and 300 μm, which is in good agreement with the distribution of SRB population as revealed by cryosection-fluorescence in situ hybridization (FISH). SRB had a higher relative abundance of 20% on the surface layer, which decreased gradually to below 3% at a depth of 400 μm. In contrast, MA mainly inhabited the inner layer of the biofilm. Their relative abundances increased from 10% to 75% at depths of 200 μm and 700 μm, respectively, from the biofilm surface layer. High-throughput pyrosequencing of 16S rRNA amplicons showed that SRB in the biofilm were mainly affiliated with five genera, Desulfobulbus, Desulfomicrobium, Desulfovibrio, Desulfatiferula, and Desulforegula, while about 90% of the MA population belonged to the genus Methanosaeta. The spatial organizations of SRB and MA revealed by pyrosequencing were consistent with the FISH results. A biofilm model was constructed to simulate the SRB and MA distributions in the anaerobic sewer biofilm. The good fit between model predictions and the experimental data indicate that the coexistence and spatial structure of SRB and MA in the biofilm resulted from the microbial types and their metabolic transformations and interactions with substrates. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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

PubMed

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

2009-11-25

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

Coupled cryoconite ecosystem structure-function relationships are revealed by comparing bacterial communities in alpine and Arctic glaciers.

PubMed

Edwards, Arwyn; Mur, Luis A J; Girdwood, Susan E; Anesio, Alexandre M; Stibal, Marek; Rassner, Sara M E; Hell, Katherina; Pachebat, Justin A; Post, Barbara; Bussell, Jennifer S; Cameron, Simon J S; Griffith, Gareth Wyn; Hodson, Andrew J; Sattler, Birgit

2014-08-01

Cryoconite holes are known as foci of microbial diversity and activity on polar glacier surfaces, but are virtually unexplored microbial habitats in alpine regions. In addition, whether cryoconite community structure reflects ecosystem functionality is poorly understood. Terminal restriction fragment length polymorphism and Fourier transform infrared metabolite fingerprinting of cryoconite from glaciers in Austria, Greenland and Svalbard demonstrated cryoconite bacterial communities are closely correlated with cognate metabolite fingerprints. The influence of bacterial-associated fatty acids and polysaccharides was inferred, underlining the importance of bacterial community structure in the properties of cryoconite. Thus, combined application of T-RFLP and FT-IR metabolite fingerprinting promises high throughput, and hence, rapid assessment of community structure-function relationships. Pyrosequencing revealed Proteobacteria were particularly abundant, with Cyanobacteria likely acting as ecosystem engineers in both alpine and Arctic cryoconite communities. However, despite these generalities, significant differences in bacterial community structures, compositions and metabolomes are found between alpine and Arctic cryoconite habitats, reflecting the impact of local and regional conditions on the challenges of thriving in glacial ecosystems. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

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

PubMed Central

Laehnemann, David; Borkhardt, Arndt

2016-01-01

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

Yeast diversity during the fermentation of Andean chicha: A comparison of high-throughput sequencing and culture-dependent approaches.

PubMed

Mendoza, Lucía M; Neef, Alexander; Vignolo, Graciela; Belloch, Carmela

2017-10-01

Diversity and dynamics of yeasts associated with the fermentation of Argentinian maize-based beverage chicha was investigated. Samples taken at different stages from two chicha productions were analyzed by culture-dependent and culture-independent methods. Five hundred and ninety six yeasts were isolated by classical microbiological methods and 16 species identified by RFLPs and sequencing of D1/D2 26S rRNA gene. Genetic typing of isolates from the dominant species, Saccharomyces cerevisiae, by PCR of delta elements revealed up to 42 different patterns. High-throughput sequencing (HTS) of D1/D2 26S rRNA gene amplicons from chicha samples detected more than one hundred yeast species and almost fifty filamentous fungi taxa. Analysis of the data revealed that yeasts dominated the fermentation, although, a significant percentage of filamentous fungi appeared in the first step of the process. Statistical analysis of results showed that very few taxa were represented by more than 1% of the reads per sample at any step of the process. S. cerevisiae represented more than 90% of the reads in the fermentative samples. Other yeast species dominated the pre-fermentative steps and abounded in fermented samples when S. cerevisiae was in percentages below 90%. Most yeasts species detected by pyrosequencing were not recovered by cultivation. In contrast, the cultivation-based methodology detected very few yeast taxa, and most of them corresponded with very few reads in the pyrosequencing analysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

PubMed Central

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. PMID:26020967

Phylogenetically Distinct Phylotypes Modulate Nitrification in a Paddy Soil

PubMed Central

Zhao, Jun; Wang, Baozhan

2015-01-01

Paddy fields represent a unique ecosystem in which regular flooding occurs, allowing for rice cultivation. However, the taxonomic identity of the microbial functional guilds that catalyze soil nitrification remains poorly understood. In this study, we provide molecular evidence for distinctly different phylotypes of nitrifying communities in a neutral paddy soil using high-throughput pyrosequencing and DNA-based stable isotope probing (SIP). Following urea addition, the levels of soil nitrate increased significantly, accompanied by an increase in the abundance of the bacterial and archaeal amoA gene in microcosms subjected to SIP (SIP microcosms) during a 56-day incubation period. High-throughput fingerprints of the total 16S rRNA genes in SIP microcosms indicated that nitrification activity positively correlated with the abundance of Nitrosospira-like ammonia-oxidizing bacteria (AOB), soil group 1.1b-like ammonia-oxidizing archaea (AOA), and Nitrospira-like nitrite-oxidizing bacteria (NOB). Pyrosequencing of 13C-labeled DNA further revealed that 13CO2 was assimilated by these functional groups to a much greater extent than by marine group 1.1a-associated AOA and Nitrobacter-like NOB. Phylogenetic analysis demonstrated that active AOB communities were closely affiliated with Nitrosospira sp. strain L115 and the Nitrosospira multiformis lineage and that the 13C-labeled AOA were related to phylogenetically distinct groups, including the moderately thermophilic “Candidatus Nitrososphaera gargensis,” uncultured fosmid 29i4, and acidophilic “Candidatus Nitrosotalea devanaterra” lineages. These results suggest that a wide variety of microorganisms were involved in soil nitrification, implying physiological diversification of soil nitrifying communities that are constantly exposed to environmental fluctuations in paddy fields. PMID:25724959

Pyrosequencing of the northern red oak (Quercus rubra L.) chloroplast genome reveals high quality polymorphisms for population management

Treesearch

Lisa W. Alexander; Keith E. Woeste

2014-01-01

Given the low intraspecific chloroplast diversity detected in northern red oak (Quercus rubra L.), more powerful genetic tools are necessary to accurately characterize Q. rubra chloroplast diversity and structure. We report the sequencing, assembly, and annotation of the chloroplast genome of northern red oak via pyrosequencing and...

Molecular ecological network analyses.

PubMed

Deng, Ye; Jiang, Yi-Huei; Yang, Yunfeng; He, Zhili; Luo, Feng; Zhou, Jizhong

2012-05-30

Understanding the interaction among different species within a community and their responses to environmental changes is a central goal in ecology. However, defining the network structure in a microbial community is very challenging due to their extremely high diversity and as-yet uncultivated status. Although recent advance of metagenomic technologies, such as high throughout sequencing and functional gene arrays, provide revolutionary tools for analyzing microbial community structure, it is still difficult to examine network interactions in a microbial community based on high-throughput metagenomics data. Here, we describe a novel mathematical and bioinformatics framework to construct ecological association networks named molecular ecological networks (MENs) through Random Matrix Theory (RMT)-based methods. Compared to other network construction methods, this approach is remarkable in that the network is automatically defined and robust to noise, thus providing excellent solutions to several common issues associated with high-throughput metagenomics data. We applied it to determine the network structure of microbial communities subjected to long-term experimental warming based on pyrosequencing data of 16 S rRNA genes. We showed that the constructed MENs under both warming and unwarming conditions exhibited topological features of scale free, small world and modularity, which were consistent with previously described molecular ecological networks. Eigengene analysis indicated that the eigengenes represented the module profiles relatively well. In consistency with many other studies, several major environmental traits including temperature and soil pH were found to be important in determining network interactions in the microbial communities examined. To facilitate its application by the scientific community, all these methods and statistical tools have been integrated into a comprehensive Molecular Ecological Network Analysis Pipeline (MENAP), which is open-accessible now (http://ieg2.ou.edu/MENA). The RMT-based molecular ecological network analysis provides powerful tools to elucidate network interactions in microbial communities and their responses to environmental changes, which are fundamentally important for research in microbial ecology and environmental microbiology.

454 pyrosequencing analysis of bacterial diversity revealed by a comparative study of soils from mining subsidence and reclamation areas.

PubMed

Li, Yuanyuan; Chen, Longqian; Wen, Hongyu; Zhou, Tianjian; Zhang, Ting; Gao, Xiali

2014-03-28

Significant alteration in the microbial community can occur across reclamation areas suffering subsidence from mining. A reclamation site undergoing fertilization practices and an adjacent coal-excavated subsidence site (sites A and B, respectively) were examined to characterize the bacterial diversity using 454 high-throughput 16S rDNA sequencing. The dominant taxonomic groups in both the sites were Proteobacteria, Acidobacteria, Bacteroidetes, Betaproteobacteria, Actinobacteria, Gammaproteobacteria, Alphaproteobacteria, Deltaproteobacteria, Chloroflexi, and Firmicutes. However, the bacterial communities' abundance, diversity, and composition differed significantly between the sites. Site A presented higher bacterial diversity and more complex community structures than site B. The majority of sequences related to Proteobacteria, Gemmatimonadetes, Chloroflexi, Nitrospirae, Firmicutes, Betaproteobacteria, Deltaproteobacteria, and Anaerolineae were from site A; whereas those related to Actinobacteria, Planctomycetes, Bacteroidetes, Verrucomicrobia, Gammaproteobacteria, Nitriliruptoria, Alphaproteobacteria, and Phycisphaerae originated from site B. The distribution of some bacterial groups and subgroups in the two sites correlated with soil properties and vegetation due to reclamation practice. Site A exhibited enriched bacterial community, soil organic matter (SOM), and total nitrogen (TN), suggesting the presence of relatively diverse microorganisms. SOM and TN were important factors shaping the underlying microbial communities. Furthermore, the specific plant functional group (legumes) was also an important factor influencing soil microbial community composition. Thus, the effectiveness of 454 pyrosequencing in analyzing soil bacterial diversity was validated and an association between land ecological system restoration, mostly mediated by microbial communities, and an improvement in soil properties in coalmining reclamation areas was suggested.

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

PubMed

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. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Response of ammonia oxidizing archaea and bacteria to decabromodiphenyl ether and copper contamination in river sediments.

PubMed

Wang, Linqiong; Li, Yi; Niu, Lihua; Zhang, Wenlong; Zhang, Huanjun; Wang, Longfei; Wang, Peifang

2018-01-01

Ammonia oxidation plays a fundamental role in river nitrogen cycling ecosystems, which is normally governed by both ammonia oxidizing archaea (AOA) and ammonia oxidizing bacteria (AOB). Co-contamination of typical emerging pollutant Polybrominated diphenyl ethers (PBDEs) and heavy metal on AOA and AOB communities in river sediments remains unknown. In this study, multiple analytical tools, including high-throughput pyrosequencing and real-time quantitative PCR (qPCR), were used to reveal the ammonia monooxygenase (AMO) activity, subunit alpha (amoA) gene abundance, and community structures of AOA and AOB in river sediments. It was found that the inhibition of AMO activities was increased with the increase of decabromodiphenyl ether (BDE 209, 1-100 mg kg -1 ) and copper (Cu, 50-500 mg kg -1 ) concentrations. Moreover, the synergic effects of BDE 209 and Cu resulted in a higher AMO activity reduction than the individual pollutant BDE 209. The AOA amoA copy number declined by 75.9% and 83.2% and AOB amoA gene abundance declined 82.8% and 90.0% at 20 and 100 mg kg -1 BDE 209 with a 100 mg kg -1 Cu co-contamination, respectively. The pyrosequencing results showed that both AOB and AOA community structures were altered, with a higher change of AOB than that of AOA. The results demonstrated that the AOB microbial community may be better adapted to BDE 209 and Cu pollution, while AOA might possess a greater capacity for stress resistance. Our study provides a better understanding of the ecotoxicological effects of heavy metal and micropollutant combined exposure on AOA and AOB in river sediments. Copyright © 2017 Elsevier Ltd. All rights reserved.

Spatial Segregation and Aggregation of Ectomycorrhizal and Root-Endophytic Fungi in the Seedlings of Two Quercus Species

PubMed Central

Yamamoto, Satoshi; Sato, Hirotoshi; Tanabe, Akifumi S.; Hidaka, Amane; Kadowaki, Kohmei; Toju, Hirokazu

2014-01-01

Diverse clades of mycorrhizal and endophytic fungi are potentially involved in competitive or facilitative interactions within host-plant roots. We investigated the potential consequences of these ecological interactions on the assembly process of root-associated fungi by examining the co-occurrence of pairs of fungi in host-plant individuals. Based on massively-parallel pyrosequencing, we analyzed the root-associated fungal community composition for each of the 249 Quercus serrata and 188 Quercus glauca seedlings sampled in a warm-temperate secondary forest in Japan. Pairs of fungi that co-occurred more or less often than expected by chance were identified based on randomization tests. The pyrosequencing analysis revealed that not only ectomycorrhizal fungi but also endophytic fungi were common in the root-associated fungal community. Intriguingly, specific pairs of these ectomycorrhizal and endophytic fungi showed spatially aggregated patterns, suggesting the existence of facilitative interactions between fungi in different functional groups. Due to the large number of fungal pairs examined, many of the observed aggregated/segregated patterns with very low P values (e.g., < 0.005) turned non-significant after the application of a multiple comparison method. However, our overall results imply that the community structures of ectomycorrhizal and endophytic fungi could influence each other through interspecific competitive/facilitative interactions in root. To test the potential of host-plants' control of fungus–fungus ecological interactions in roots, we further examined whether the aggregated/segregated patterns could vary depending on the identity of host plant species. Potentially due to the physiological properties shared between the congeneric host plant species, the sign of hosts' control was not detected in the present study. The pyrosequencing-based randomization analyses shown in this study provide a platform of the high-throughput investigation of fungus–fungus interactions in plant root systems. PMID:24801150

Enhanced phenol removal in an innovative lignite activated coke-assisted biological process.

PubMed

Zhang, Chen; Li, Jianfeng; Cheng, Fangqin; Liu, Yu

2018-07-01

In this study, a lignite activated coke (LAC)-assisted activated sludge (AS) process was developed for enhancing biodegradation of phenol, while the effects of LAC on sludge properties and microbial community structure were investigated. It was found that more than 90% of phenol was removed within 1 h in the LAC/AS, which was 3 times higher than the conventional AS process. Moreover, the floc size and settleability were also significantly improved in the LAC/AS. These results suggested that LAC could serve as the nucleating agent to promote the formation of compact floc, which was beneficial for toxicity mitigation and system stability. The microbial community analysis by 16S high-throughput pyrosequencing technology further revealed a more abundant bacterial richness and diversity in the LAC/AS process loaded with phenol, while some phenol degraders, such as Propionibacteriaceae were enriched. Engineering implications further suggests the LAC-assisted AS process is technically sound and economically viable. Copyright © 2018 Elsevier Ltd. All rights reserved.

High-throughput pyrosequencing used for the discovery of a novel cellulase from a thermophilic cellulose-degrading microbial consortium.

PubMed

Zhao, Chao; Chu, Yanan; Li, Yanhong; Yang, Chengfeng; Chen, Yuqing; Wang, Xumin; Liu, Bin

2017-01-01

To analyze the microbial diversity and gene content of a thermophilic cellulose-degrading consortium from hot springs in Xiamen, China using 454 pyrosequencing for discovering cellulolytic enzyme resources. A thermophilic cellulose-degrading consortium, XM70 that was isolated from a hot spring, used sugarcane bagasse as sole carbon and energy source. DNA sequencing of the XM70 sample resulted in 349,978 reads with an average read length of 380 bases, accounting for 133,896,867 bases of sequence information. The characterization of sequencing reads and assembled contigs revealed that most microbes were derived from four phyla: Geobacillus (Firmicutes), Thermus, Bacillus, and Anoxybacillus. Twenty-eight homologous genes belonging to 15 glycoside hydrolase families were detected, including several cellulase genes. A novel hot spring metagenome-derived thermophilic cellulase was expressed and characterized. The application value of thermostable sugarcane bagasse-degrading enzymes is shown for production of cellulosic biofuel. The practical power of using a short-read-based metagenomic approach for harvesting novel microbial genes is also demonstrated.

MSP-HTPrimer: a high-throughput primer design tool to improve assay design for DNA methylation analysis in epigenetics.

PubMed

Pandey, Ram Vinay; Pulverer, Walter; Kallmeyer, Rainer; Beikircher, Gabriel; Pabinger, Stephan; Kriegner, Albert; Weinhäusel, Andreas

2016-01-01

Bisulfite (BS) conversion-based and methylation-sensitive restriction enzyme (MSRE)-based PCR methods have been the most commonly used techniques for locus-specific DNA methylation analysis. However, both methods have advantages and limitations. Thus, an integrated approach would be extremely useful to quantify the DNA methylation status successfully with great sensitivity and specificity. Designing specific and optimized primers for target regions is the most critical and challenging step in obtaining the adequate DNA methylation results using PCR-based methods. Currently, no integrated, optimized, and high-throughput methylation-specific primer design software methods are available for both BS- and MSRE-based methods. Therefore an integrated, powerful, and easy-to-use methylation-specific primer design pipeline with great accuracy and success rate will be very useful. We have developed a new web-based pipeline, called MSP-HTPrimer, to design primers pairs for MSP, BSP, pyrosequencing, COBRA, and MSRE assays on both genomic strands. First, our pipeline converts all target sequences into bisulfite-treated templates for both forward and reverse strand and designs all possible primer pairs, followed by filtering for single nucleotide polymorphisms (SNPs) and known repeat regions. Next, each primer pairs are annotated with the upstream and downstream RefSeq genes, CpG island, and cut sites (for COBRA and MSRE). Finally, MSP-HTPrimer selects specific primers from both strands based on custom and user-defined hierarchical selection criteria. MSP-HTPrimer produces a primer pair summary output table in TXT and HTML format for display and UCSC custom tracks for resulting primer pairs in GTF format. MSP-HTPrimer is an integrated, web-based, and high-throughput pipeline and has no limitation on the number and size of target sequences and designs MSP, BSP, pyrosequencing, COBRA, and MSRE assays. It is the only pipeline, which automatically designs primers on both genomic strands to increase the success rate. It is a standalone web-based pipeline, which is fully configured within a virtual machine and thus can be readily used without any configuration. We have experimentally validated primer pairs designed by our pipeline and shown a very high success rate of primer pairs: out of 66 BSP primer pairs, 63 were successfully validated without any further optimization step and using the same qPCR conditions. The MSP-HTPrimer pipeline is freely available from http://sourceforge.net/p/msp-htprimer.

Identification of Methylated Genes Associated with Aggressive Bladder Cancer

PubMed Central

Marsit, Carmen J.; Houseman, E. Andres; Christensen, Brock C.; Gagne, Luc; Wrensch, Margaret R.; Nelson, Heather H.; Wiemels, Joseph; Zheng, Shichun; Wiencke, John K.; Andrew, Angeline S.; Schned, Alan R.; Karagas, Margaret R.; Kelsey, Karl T.

2010-01-01

Approximately 500,000 individuals diagnosed with bladder cancer in the U.S. require routine cystoscopic follow-up to monitor for disease recurrences or progression, resulting in over $2 billion in annual expenditures. Identification of new diagnostic and monitoring strategies are clearly needed, and markers related to DNA methylation alterations hold great promise due to their stability, objective measurement, and known associations with the disease and with its clinical features. To identify novel epigenetic markers of aggressive bladder cancer, we utilized a high-throughput DNA methylation bead-array in two distinct population-based series of incident bladder cancer (n = 73 and n = 264, respectively). We then validated the association between methylation of these candidate loci with tumor grade in a third population (n = 245) through bisulfite pyrosequencing of candidate loci. Array based analyses identified 5 loci for further confirmation with bisulfite pyrosequencing. We identified and confirmed that increased promoter methylation of HOXB2 is significantly and independently associated with invasive bladder cancer and methylation of HOXB2, KRT13 and FRZB together significantly predict high-grade non-invasive disease. Methylation of these genes may be useful as clinical markers of the disease and may point to genes and pathways worthy of additional examination as novel targets for therapeutic treatment. PMID:20808801

Identification of methylated genes associated with aggressive bladder cancer.

PubMed

Marsit, Carmen J; Houseman, E Andres; Christensen, Brock C; Gagne, Luc; Wrensch, Margaret R; Nelson, Heather H; Wiemels, Joseph; Zheng, Shichun; Wiencke, John K; Andrew, Angeline S; Schned, Alan R; Karagas, Margaret R; Kelsey, Karl T

2010-08-23

Approximately 500,000 individuals diagnosed with bladder cancer in the U.S. require routine cystoscopic follow-up to monitor for disease recurrences or progression, resulting in over $2 billion in annual expenditures. Identification of new diagnostic and monitoring strategies are clearly needed, and markers related to DNA methylation alterations hold great promise due to their stability, objective measurement, and known associations with the disease and with its clinical features. To identify novel epigenetic markers of aggressive bladder cancer, we utilized a high-throughput DNA methylation bead-array in two distinct population-based series of incident bladder cancer (n = 73 and n = 264, respectively). We then validated the association between methylation of these candidate loci with tumor grade in a third population (n = 245) through bisulfite pyrosequencing of candidate loci. Array based analyses identified 5 loci for further confirmation with bisulfite pyrosequencing. We identified and confirmed that increased promoter methylation of HOXB2 is significantly and independently associated with invasive bladder cancer and methylation of HOXB2, KRT13 and FRZB together significantly predict high-grade non-invasive disease. Methylation of these genes may be useful as clinical markers of the disease and may point to genes and pathways worthy of additional examination as novel targets for therapeutic treatment.

Bacterial community comparisons by taxonomy-supervised analysis independent of sequence alignment and clustering

PubMed Central

Sul, Woo Jun; Cole, James R.; Jesus, Ederson da C.; Wang, Qiong; Farris, Ryan J.; Fish, Jordan A.; Tiedje, James M.

2011-01-01

High-throughput sequencing of 16S rRNA genes has increased our understanding of microbial community structure, but now even higher-throughput methods to the Illumina scale allow the creation of much larger datasets with more samples and orders-of-magnitude more sequences that swamp current analytic methods. We developed a method capable of handling these larger datasets on the basis of assignment of sequences into an existing taxonomy using a supervised learning approach (taxonomy-supervised analysis). We compared this method with a commonly used clustering approach based on sequence similarity (taxonomy-unsupervised analysis). We sampled 211 different bacterial communities from various habitats and obtained ∼1.3 million 16S rRNA sequences spanning the V4 hypervariable region by pyrosequencing. Both methodologies gave similar ecological conclusions in that β-diversity measures calculated by using these two types of matrices were significantly correlated to each other, as were the ordination configurations and hierarchical clustering dendrograms. In addition, our taxonomy-supervised analyses were also highly correlated with phylogenetic methods, such as UniFrac. The taxonomy-supervised analysis has the advantages that it is not limited by the exhaustive computation required for the alignment and clustering necessary for the taxonomy-unsupervised analysis, is more tolerant of sequencing errors, and allows comparisons when sequences are from different regions of the 16S rRNA gene. With the tremendous expansion in 16S rRNA data acquisition underway, the taxonomy-supervised approach offers the potential to provide more rapid and extensive community comparisons across habitats and samples. PMID:21873204

The Analysis of a Microbial Community in the UV/O3-Anaerobic/Aerobic Integrated Process for Petrochemical Nanofiltration Concentrate (NFC) Treatment by 454-Pyrosequencing.

PubMed

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.

The Analysis of a Microbial Community in the UV/O3-Anaerobic/Aerobic Integrated Process for Petrochemical Nanofiltration Concentrate (NFC) Treatment by 454-Pyrosequencing

PubMed Central

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

Droplet-based pyrosequencing using digital microfluidics.

PubMed

Boles, Deborah J; Benton, Jonathan L; Siew, Germaine J; Levy, Miriam H; Thwar, Prasanna K; Sandahl, Melissa A; Rouse, Jeremy L; Perkins, Lisa C; Sudarsan, Arjun P; Jalili, Roxana; Pamula, Vamsee K; Srinivasan, Vijay; Fair, Richard B; Griffin, Peter B; Eckhardt, Allen E; Pollack, Michael G

2011-11-15

The feasibility of implementing pyrosequencing chemistry within droplets using electrowetting-based digital microfluidics is reported. An array of electrodes patterned on a printed-circuit board was used to control the formation, transportation, merging, mixing, and splitting of submicroliter-sized droplets contained within an oil-filled chamber. A three-enzyme pyrosequencing protocol was implemented in which individual droplets contained enzymes, deoxyribonucleotide triphosphates (dNTPs), and DNA templates. The DNA templates were anchored to magnetic beads which enabled them to be thoroughly washed between nucleotide additions. Reagents and protocols were optimized to maximize signal over background, linearity of response, cycle efficiency, and wash efficiency. As an initial demonstration of feasibility, a portion of a 229 bp Candida parapsilosis template was sequenced using both a de novo protocol and a resequencing protocol. The resequencing protocol generated over 60 bp of sequence with 100% sequence accuracy based on raw pyrogram levels. Excellent linearity was observed for all of the homopolymers (two, three, or four nucleotides) contained in the C. parapsilosis sequence. With improvements in microfluidic design it is expected that longer reads, higher throughput, and improved process integration (i.e., "sample-to-sequence" capability) could eventually be achieved using this low-cost platform.

Droplet-Based Pyrosequencing Using Digital Microfluidics

PubMed Central

Boles, Deborah J.; Benton, Jonathan L.; Siew, Germaine J.; Levy, Miriam H.; Thwar, Prasanna K.; Sandahl, Melissa A.; Rouse, Jeremy L.; Perkins, Lisa C.; Sudarsan, Arjun P.; Jalili, Roxana; Pamula, Vamsee K.; Srinivasan, Vijay; Fair, Richard B.; Griffin, Peter B.; Eckhardt, Allen E.; Pollack, Michael G.

2013-01-01

The feasibility of implementing pyrosequencing chemistry within droplets using electrowetting-based digital microfluidics is reported. An array of electrodes patterned on a printed-circuit board was used to control the formation, transportation, merging, mixing, and splitting of submicroliter-sized droplets contained within an oil-filled chamber. A three-enzyme pyrosequencing protocol was implemented in which individual droplets contained enzymes, deoxyribonucleotide triphosphates (dNTPs), and DNA templates. The DNA templates were anchored to magnetic beads which enabled them to be thoroughly washed between nucleotide additions. Reagents and protocols were optimized to maximize signal over background, linearity of response, cycle efficiency, and wash efficiency. As an initial demonstration of feasibility, a portion of a 229 bp Candida parapsilosis template was sequenced using both a de novo protocol and a resequencing protocol. The resequencing protocol generated over 60 bp of sequence with 100% sequence accuracy based on raw pyrogram levels. Excellent linearity was observed for all of the homopolymers (two, three, or four nucleotides) contained in the C. parapsilosis sequence. With improvements in microfluidic design it is expected that longer reads, higher throughput, and improved process integration (i.e., “sample-to-sequence” capability) could eventually be achieved using this low-cost platform. PMID:21932784

The microbiota of marketed processed edible insects as revealed by high-throughput sequencing.

PubMed

Garofalo, Cristiana; Osimani, Andrea; Milanović, Vesna; Taccari, Manuela; Cardinali, Federica; Aquilanti, Lucia; Riolo, Paola; Ruschioni, Sara; Isidoro, Nunzio; Clementi, Francesca

2017-04-01

Entomophagy has been linked to nutritional, economic, social and ecological benefits. However, scientific studies on the potential safety risks in eating edible insects need to be carried out for legislators, markets and consumers. In this context, the microbiota of edible insects deserves to be deeply investigated. The aim of this study was to elucidate the microbial species occurring in some processed marketed edible insects, namely powdered small crickets, whole dried small crickets (Acheta domesticus), whole dried locusts (Locusta migratoria), and whole dried mealworm larvae (Tenebrio molitor), through culture-dependent (classical microbiological analyses) and -independent methods (pyrosequencing). A great bacterial diversity and variation among insects was seen. Relatively low counts of total mesophilic aerobes, Enterobacteriaceae, lactic acid bacteria, Clostridium perfringens spores, yeasts and moulds in all of the studied insect batches were found. Furthermore, the presence of several gut-associated bacteria, some of which may act as opportunistic pathogens in humans, were found through pyrosequencing. Food spoilage bacteria were also identified, as well as Spiroplasma spp. in mealworm larvae, which has been found to be related to neurodegenerative diseases in animals and humans. Although viable pathogens such as Salmonella spp. and Listeria monocytogenes were not detected, the presence of Listeria spp., Staphylococcus spp., Clostridium spp. and Bacillus spp. (with low abundance) was also found through pyrosequencing. The results of this study contribute to the elucidation of the microbiota associated with edible insects and encourage further studies aimed to evaluate the influence of rearing and processing conditions on that microbiota. Copyright © 2016 Elsevier Ltd. All rights reserved.

Distinct Soil Bacterial Communities Revealed under a Diversely Managed Agroecosystem

PubMed Central

Shange, Raymon S.; Ankumah, Ramble O.; Ibekwe, Abasiofiok M.; Zabawa, Robert; Dowd, Scot E.

2012-01-01

Land-use change and management practices are normally enacted to manipulate environments to improve conditions that relate to production, remediation, and accommodation. However, their effect on the soil microbial community and their subsequent influence on soil function is still difficult to quantify. Recent applications of molecular techniques to soil biology, especially the use of 16S rRNA, are helping to bridge this gap. In this study, the influence of three land-use systems within a demonstration farm were evaluated with a view to further understand how these practices may impact observed soil bacterial communities. Replicate soil samples collected from the three land-use systems (grazed pine forest, cultivated crop, and grazed pasture) on a single soil type. High throughput 16S rRNA gene pyrosequencing was used to generate sequence datasets. The different land use systems showed distinction in the structure of their bacterial communities with respect to the differences detected in cluster analysis as well as diversity indices. Specific taxa, particularly Actinobacteria, Acidobacteria, and classes of Proteobacteria, showed significant shifts across the land-use strata. Families belonging to these taxa broke with notions of copio- and oligotrphy at the class level, as many of the less abundant groups of families of Actinobacteria showed a propensity for soil environments with reduced carbon/nutrient availability. Orders Actinomycetales and Solirubrobacterales showed their highest abundance in the heavily disturbed cultivated system despite the lowest soil organic carbon (SOC) values across the site. Selected soil properties ([SOC], total nitrogen [TN], soil texture, phosphodiesterase [PD], alkaline phosphatase [APA], acid phosphatase [ACP] activity, and pH) also differed significantly across land-use regimes, with SOM, PD, and pH showing variation consistent with shifts in community structure and composition. These results suggest that use of pyrosequencing along with traditional analysis of soil physiochemical properties may provide insight into the ecology of descending taxonomic groups in bacterial communities. PMID:22844402

Distinct soil bacterial communities revealed under a diversely managed agroecosystem.

PubMed

Shange, Raymon S; Ankumah, Ramble O; Ibekwe, Abasiofiok M; Zabawa, Robert; Dowd, Scot E

2012-01-01

Land-use change and management practices are normally enacted to manipulate environments to improve conditions that relate to production, remediation, and accommodation. However, their effect on the soil microbial community and their subsequent influence on soil function is still difficult to quantify. Recent applications of molecular techniques to soil biology, especially the use of 16S rRNA, are helping to bridge this gap. In this study, the influence of three land-use systems within a demonstration farm were evaluated with a view to further understand how these practices may impact observed soil bacterial communities. Replicate soil samples collected from the three land-use systems (grazed pine forest, cultivated crop, and grazed pasture) on a single soil type. High throughput 16S rRNA gene pyrosequencing was used to generate sequence datasets. The different land use systems showed distinction in the structure of their bacterial communities with respect to the differences detected in cluster analysis as well as diversity indices. Specific taxa, particularly Actinobacteria, Acidobacteria, and classes of Proteobacteria, showed significant shifts across the land-use strata. Families belonging to these taxa broke with notions of copio- and oligotrphy at the class level, as many of the less abundant groups of families of Actinobacteria showed a propensity for soil environments with reduced carbon/nutrient availability. Orders Actinomycetales and Solirubrobacterales showed their highest abundance in the heavily disturbed cultivated system despite the lowest soil organic carbon (SOC) values across the site. Selected soil properties ([SOC], total nitrogen [TN], soil texture, phosphodiesterase [PD], alkaline phosphatase [APA], acid phosphatase [ACP] activity, and pH) also differed significantly across land-use regimes, with SOM, PD, and pH showing variation consistent with shifts in community structure and composition. These results suggest that use of pyrosequencing along with traditional analysis of soil physiochemical properties may provide insight into the ecology of descending taxonomic groups in bacterial communities.

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

NASA Astrophysics Data System (ADS)

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

2017-12-01

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

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

PubMed

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

2017-01-01

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

High-throughput Methods Redefine the Rumen Microbiome and Its Relationship with Nutrition and Metabolism

PubMed Central

McCann, Joshua C.; Wickersham, Tryon A.; Loor, Juan J.

2014-01-01

Diversity in the forestomach microbiome is one of the key features of ruminant animals. The diverse microbial community adapts to a wide array of dietary feedstuffs and management strategies. Understanding rumen microbiome composition, adaptation, and function has global implications ranging from climatology to applied animal production. Classical knowledge of rumen microbiology was based on anaerobic, culture-dependent methods. Next-generation sequencing and other molecular techniques have uncovered novel features of the rumen microbiome. For instance, pyrosequencing of the 16S ribosomal RNA gene has revealed the taxonomic identity of bacteria and archaea to the genus level, and when complemented with barcoding adds multiple samples to a single run. Whole genome shotgun sequencing generates true metagenomic sequences to predict the functional capability of a microbiome, and can also be used to construct genomes of isolated organisms. Integration of high-throughput data describing the rumen microbiome with classic fermentation and animal performance parameters has produced meaningful advances and opened additional areas for study. In this review, we highlight recent studies of the rumen microbiome in the context of cattle production focusing on nutrition, rumen development, animal efficiency, and microbial function. PMID:24940050

Diversity and functions of bacterial community in drinking water biofilms revealed by high-throughput sequencing

PubMed Central

Chao, Yuanqing; Mao, Yanping; Wang, Zhiping; Zhang, Tong

2015-01-01

The development of biofilms in drinking water (DW) systems may cause various problems to water quality. To investigate the community structure of biofilms on different pipe materials and the global/specific metabolic functions of DW biofilms, PCR-based 454 pyrosequencing data for 16S rRNA genes and Illumina metagenomic data were generated and analysed. Considerable differences in bacterial diversity and taxonomic structure were identified between biofilms formed on stainless steel and biofilms formed on plastics, indicating that the metallic materials facilitate the formation of higher diversity biofilms. Moreover, variations in several dominant genera were observed during biofilm formation. Based on PCA analysis, the global functions in the DW biofilms were similar to other DW metagenomes. Beyond the global functions, the occurrences and abundances of specific protective genes involved in the glutathione metabolism, the SoxRS system, the OxyR system, RpoS regulated genes, and the production/degradation of extracellular polymeric substances were also evaluated. A near-complete and low-contamination draft genome was constructed from the metagenome of the DW biofilm, based on the coverage and tetranucleotide frequencies, and identified as a Bradyrhizobiaceae-like bacterium according to a phylogenetic analysis. Our findings provide new insight into DW biofilms, especially in terms of their metabolic functions. PMID:26067561

Changes in the Bacterial Community of Soil from a Neutral Mine Drainage Channel

PubMed Central

Pereira, Letícia Bianca; Vicentini, Renato; Ottoboni, Laura M. M.

2014-01-01

Mine drainage is an important environmental disturbance that affects the chemical and biological components in natural resources. However, little is known about the effects of neutral mine drainage on the soil bacteria community. Here, a high-throughput 16S rDNA pyrosequencing approach was used to evaluate differences in composition, structure, and diversity of bacteria communities in samples from a neutral drainage channel, and soil next to the channel, at the Sossego copper mine in Brazil. Advanced statistical analyses were used to explore the relationships between the biological and chemical data. The results showed that the neutral mine drainage caused changes in the composition and structure of the microbial community, but not in its diversity. The Deinococcus/Thermus phylum, especially the Meiothermus genus, was in large part responsible for the differences between the communities, and was positively associated with the presence of copper and other heavy metals in the environmental samples. Other important parameters that influenced the bacterial diversity and composition were the elements potassium, sodium, nickel, and zinc, as well as pH. The findings contribute to the understanding of bacterial diversity in soils impacted by neutral mine drainage, and demonstrate that heavy metals play an important role in shaping the microbial population in mine environments. PMID:24796430

Diversity and functions of bacterial community in drinking water biofilms revealed by high-throughput sequencing

NASA Astrophysics Data System (ADS)

Chao, Yuanqing; Mao, Yanping; Wang, Zhiping; Zhang, Tong

2015-06-01

The development of biofilms in drinking water (DW) systems may cause various problems to water quality. To investigate the community structure of biofilms on different pipe materials and the global/specific metabolic functions of DW biofilms, PCR-based 454 pyrosequencing data for 16S rRNA genes and Illumina metagenomic data were generated and analysed. Considerable differences in bacterial diversity and taxonomic structure were identified between biofilms formed on stainless steel and biofilms formed on plastics, indicating that the metallic materials facilitate the formation of higher diversity biofilms. Moreover, variations in several dominant genera were observed during biofilm formation. Based on PCA analysis, the global functions in the DW biofilms were similar to other DW metagenomes. Beyond the global functions, the occurrences and abundances of specific protective genes involved in the glutathione metabolism, the SoxRS system, the OxyR system, RpoS regulated genes, and the production/degradation of extracellular polymeric substances were also evaluated. A near-complete and low-contamination draft genome was constructed from the metagenome of the DW biofilm, based on the coverage and tetranucleotide frequencies, and identified as a Bradyrhizobiaceae-like bacterium according to a phylogenetic analysis. Our findings provide new insight into DW biofilms, especially in terms of their metabolic functions.

Diversity and functions of bacterial community in drinking water biofilms revealed by high-throughput sequencing.

PubMed

Chao, Yuanqing; Mao, Yanping; Wang, Zhiping; Zhang, Tong

2015-06-12

The development of biofilms in drinking water (DW) systems may cause various problems to water quality. To investigate the community structure of biofilms on different pipe materials and the global/specific metabolic functions of DW biofilms, PCR-based 454 pyrosequencing data for 16S rRNA genes and Illumina metagenomic data were generated and analysed. Considerable differences in bacterial diversity and taxonomic structure were identified between biofilms formed on stainless steel and biofilms formed on plastics, indicating that the metallic materials facilitate the formation of higher diversity biofilms. Moreover, variations in several dominant genera were observed during biofilm formation. Based on PCA analysis, the global functions in the DW biofilms were similar to other DW metagenomes. Beyond the global functions, the occurrences and abundances of specific protective genes involved in the glutathione metabolism, the SoxRS system, the OxyR system, RpoS regulated genes, and the production/degradation of extracellular polymeric substances were also evaluated. A near-complete and low-contamination draft genome was constructed from the metagenome of the DW biofilm, based on the coverage and tetranucleotide frequencies, and identified as a Bradyrhizobiaceae-like bacterium according to a phylogenetic analysis. Our findings provide new insight into DW biofilms, especially in terms of their metabolic functions.

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

PubMed

Liu, Yun; Grossman, Jeffrey C

2014-12-10

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

Mineral Ecology: Surface Specific Colonization and Geochemical Drivers of Biofilm Accumulation, Composition, and Phylogeny

PubMed Central

Jones, Aaron A.; Bennett, Philip C.

2017-01-01

This study tests the hypothesis that surface composition influences microbial community structure and growth of biofilms. We used laboratory biofilm reactors (inoculated with a diverse subsurface community) to explore the phylogenetic and taxonomic variability in microbial communities as a function of surface type (carbonate, silicate, aluminosilicate), media pH, and carbon and phosphate availability. Using high-throughput pyrosequencing, we found that surface type significantly controlled ~70–90% of the variance in phylogenetic diversity regardless of environmental pressures. Consistent patterns also emerged in the taxonomy of specific guilds (sulfur-oxidizers/reducers, Gram-positives, acidophiles) due to variations in media chemistry. Media phosphate availability was a key property associated with variation in phylogeny and taxonomy of whole reactors and was negatively correlated with biofilm accumulation and α-diversity (species richness and evenness). However, mineral-bound phosphate limitations were correlated with less biofilm. Carbon added to the media was correlated with a significant increase in biofilm accumulation and overall α-diversity. Additionally, planktonic communities were phylogenetically distant from those in biofilms. All treatments harbored structurally (taxonomically and phylogenetically) distinct microbial communities. Selective advantages within each treatment encouraged growth and revealed the presence of hundreds of additional operational taxonomix units (OTU), representing distinct consortiums of microorganisms. Ultimately, these results provide evidence that mineral/rock composition significantly influences microbial community structure, diversity, membership, phylogenetic variability, and biofilm growth in subsurface communities. PMID:28400754

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

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

Liu, Y; Grossman, JC

2014-12-01

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

Lentinula edodes-derived polysaccharide alters the spatial structure of gut microbiota in mice.

PubMed

Xu, Xiaofei; Zhang, Xuewu

2015-01-01

Lentinula edodes-derived polysaccharides possess many therapeutic characteristics, including anti-tumor and immuno-modulation. The gut microbes play a critical role in modulation of immune function. However, the impact of Lentinula edodes-derived polysaccharides on the gut microbes have not yet been explored. In this study, high-throughput pyrosequencing technique was employed to investigate the effects of a new heteropolysaccharide L2 from Lentinula edodes on microbiota diversity and composition of small intestine, cecum, colon and distal end of colon (feces) in mice. The results demonstrated that along mouse intestine the microbiota exhibit distinctly different space distribution. L2 treatment reduced the diversity and evenness of gut microbiota along the intestine, especially in the cecum and colon. In the fecal microbial communities, the decrease of Bacteroidetes by significantly increasing Proteobacteria were observed, which were characterized by the increased Helicobacteraceae and reduced S24-7 at family level. Some OTUs, corresponding to Bacteroides acidifaciens, Alistipes and Helicobacter suncus, were found to be significantly increased in L2 treated-mice. In particular, 4 phyla Chloroflexi, Gemmatimonadetes, Nitrospirae and Planctomycetes are exclusively present in L2-treated mice. This is helpful for further demonstrating healthy action mechanism of Lentinula edodes-derived polysaccharide L2.

Sensitivity and accuracy of high-throughput metabarcoding methods for early detection of invasive fish species

NASA Astrophysics Data System (ADS)

Hatzenbuhler, Chelsea; Kelly, John R.; Martinson, John; Okum, Sara; Pilgrim, Erik

2017-04-01

High-throughput DNA metabarcoding has gained recognition as a potentially powerful tool for biomonitoring, including early detection of aquatic invasive species (AIS). DNA based techniques are advancing, but our understanding of the limits to detection for metabarcoding complex samples is inadequate. For detecting AIS at an early stage of invasion when the species is rare, accuracy at low detection limits is key. To evaluate the utility of metabarcoding in future fish community monitoring programs, we conducted several experiments to determine the sensitivity and accuracy of routine metabarcoding methods. Experimental mixes used larval fish tissue from multiple “common” species spiked with varying proportions of tissue from an additional “rare” species. Pyrosequencing of genetic marker, COI (cytochrome c oxidase subunit I) and subsequent sequence data analysis provided experimental evidence of low-level detection of the target “rare” species at biomass percentages as low as 0.02% of total sample biomass. Limits to detection varied interspecifically and were susceptible to amplification bias. Moreover, results showed some data processing methods can skew sequence-based biodiversity measurements from corresponding relative biomass abundances and increase false absences. We suggest caution in interpreting presence/absence and relative abundance in larval fish assemblages until metabarcoding methods are optimized for accuracy and precision.

Rapid phylogenetic dissection of prokaryotic community structure in tidal flat using pyrosequencing.

PubMed

Kim, Bong-Soo; Kim, Byung Kwon; Lee, Jae-Hak; Kim, Myungjin; Lim, Young Woon; Chun, Jongsik

2008-08-01

Dissection of prokaryotic community structure is prerequisite to understand their ecological roles. Various methods are available for such a purpose which amplification and sequencing of 16S rRNA genes gained its popularity. However, conventional methods based on Sanger sequencing technique require cloning process prior to sequencing, and are expensive and labor-intensive. We investigated prokaryotic community structure in tidal flat sediments, Korea, using pyrosequencing and a subsequent automated bioinformatic pipeline for the rapid and accurate taxonomic assignment of each amplicon. The combination of pyrosequencing and bioinformatic analysis showed that bacterial and archaeal communities were more diverse than previously reported in clone library studies. Pyrosequencing analysis revealed 21 bacterial divisions and 37 candidate divisions. Proteobacteria was the most abundant division in the bacterial community, of which Gamma-and Delta-Proteobacteria were the most abundant. Similarly, 4 archaeal divisions were found in tidal flat sediments. Euryarchaeota was the most abundant division in the archaeal sequences, which were further divided into 8 classes and 11 unclassified euryarchaeota groups. The system developed here provides a simple, in-depth and automated way of dissecting a prokaryotic community structure without extensive pretreatment such as cloning.

New insights into Acinetobacter baumannii pathogenesis revealed by high-density pyrosequencing and transposon mutagenesis.

PubMed

Smith, Michael G; Gianoulis, Tara A; Pukatzki, Stefan; Mekalanos, John J; Ornston, L Nicholas; Gerstein, Mark; Snyder, Michael

2007-03-01

Acinetobacter baumannii has emerged as an important and problematic human pathogen as it is the causative agent of several types of infections including pneumonia, meningitis, septicemia, and urinary tract infections. We explored the pathogenic content of this harmful pathogen using a combination of DNA sequencing and insertional mutagenesis. The genome of this organism was sequenced using a strategy involving high-density pyrosequencing, a novel, rapid method of high-throughput sequencing. Excluding the rDNA repeats, the assembled genome is 3,976,746 base pairs (bp) and has 3830 ORFs. A significant fraction of ORFs (17.2%) are located in 28 putative alien islands, indicating that the genome has acquired a large amount of foreign DNA. Consistent with its role in pathogenesis, a remarkable number of the islands (16) contain genes implicated in virulence, indicating the organism devotes a considerable portion of its genes to pathogenesis. The largest island contains elements homologous to the Legionella/Coxiella Type IV secretion apparatus. Type IV secretion systems have been demonstrated to be important for virulence in other organisms and thus are likely to help mediate pathogenesis of A. baumannii. Insertional mutagenesis generated avirulent isolates of A. baumannii and verified that six of the islands contain virulence genes, including two novel islands containing genes that lacked homology with others in the databases. The DNA sequencing approach described in this study allows the rapid elucidation of the DNA sequence of any microbe and, when combined with genetic screens, can identify many novel genes important for microbial pathogenesis.

A high-throughput microfluidic dental plaque biofilm system to visualize and quantify the effect of antimicrobials

PubMed Central

Nance, William C.; Dowd, Scot E.; Samarian, Derek; Chludzinski, Jeffrey; Delli, Joseph; Battista, John; Rickard, Alexander H.

2013-01-01

Objectives Few model systems are amenable to developing multi-species biofilms in parallel under environmentally germane conditions. This is a problem when evaluating the potential real-world effectiveness of antimicrobials in the laboratory. One such antimicrobial is cetylpyridinium chloride (CPC), which is used in numerous over-the-counter oral healthcare products. The aim of this work was to develop a high-throughput microfluidic system that is combined with a confocal laser scanning microscope (CLSM) to quantitatively evaluate the effectiveness of CPC against oral multi-species biofilms grown in human saliva. Methods Twenty-four-channel BioFlux microfluidic plates were inoculated with pooled human saliva and fed filter-sterilized saliva for 20 h at 37°C. The bacterial diversity of the biofilms was evaluated by bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP). The antimicrobial/anti-biofilm effect of CPC (0.5%–0.001% w/v) was examined using Live/Dead stain, CLSM and 3D imaging software. Results The analysis of biofilms by bTEFAP demonstrated that they contained genera typically found in human dental plaque. These included Aggregatibacter, Fusobacterium, Neisseria, Porphyromonas, Streptococcus and Veillonella. Using Live/Dead stain, clear gradations in killing were observed when the biofilms were treated with CPC between 0.5% and 0.001% w/v. At 0.5% (w/v) CPC, 90% of the total signal was from dead/damaged cells. Below this concentration range, less killing was observed. In the 0.5%–0.05% (w/v) range CPC penetration/killing was greatest and biofilm thickness was significantly reduced. Conclusions This work demonstrates the utility of a high-throughput microfluidic–CLSM system to grow multi-species oral biofilms, which are compositionally similar to naturally occurring biofilms, to assess the effectiveness of antimicrobials. PMID:23800904

High-throughput crystal-optimization strategies in the South Paris Yeast Structural Genomics Project: one size fits all?

PubMed

Leulliot, Nicolas; Trésaugues, Lionel; Bremang, Michael; Sorel, Isabelle; Ulryck, Nathalie; Graille, Marc; Aboulfath, Ilham; Poupon, Anne; Liger, Dominique; Quevillon-Cheruel, Sophie; Janin, Joël; van Tilbeurgh, Herman

2005-06-01

Crystallization has long been regarded as one of the major bottlenecks in high-throughput structural determination by X-ray crystallography. Structural genomics projects have addressed this issue by using robots to set up automated crystal screens using nanodrop technology. This has moved the bottleneck from obtaining the first crystal hit to obtaining diffraction-quality crystals, as crystal optimization is a notoriously slow process that is difficult to automatize. This article describes the high-throughput optimization strategies used in the Yeast Structural Genomics project, with selected successful examples.

Automated crystallographic system for high-throughput protein structure determination.

PubMed

Brunzelle, Joseph S; Shafaee, Padram; Yang, Xiaojing; Weigand, Steve; Ren, Zhong; Anderson, Wayne F

2003-07-01

High-throughput structural genomic efforts require software that is highly automated, distributive and requires minimal user intervention to determine protein structures. Preliminary experiments were set up to test whether automated scripts could utilize a minimum set of input parameters and produce a set of initial protein coordinates. From this starting point, a highly distributive system was developed that could determine macromolecular structures at a high throughput rate, warehouse and harvest the associated data. The system uses a web interface to obtain input data and display results. It utilizes a relational database to store the initial data needed to start the structure-determination process as well as generated data. A distributive program interface administers the crystallographic programs which determine protein structures. Using a test set of 19 protein targets, 79% were determined automatically.

High-throughput sequencing for the detection of the bacterial and fungal diversity in Mongolian naturally fermented cow's milk in Russia.

PubMed

Liu, Wenjun; Zheng, Yi; Kwok, Lai-Yu; Sun, Zhihong; Zhang, Jiachao; Guo, Zhuang; Hou, Qiangchuan; Menhe, Bilige; Zhang, Heping

2015-02-22

Traditional fermented dairy products are major components of the typical Mongolian diet since ancient times. However, almost all the previous studies on the microbial composition of traditional Mongolian fermented dairy products analyzed food samples from the Chinese Mongolian region and Mongolia but not the Russian Mongolian region. In this study, the bacterial and fungal community diversity of nineteen naturally fermented cow's milk (NFCM) samples from local Mongolian families residing in Kalmykia and Chita of Russia was investigated with pyrosequencing. Firmicutes and Ascomycota were the predominant phyla respectively for bacteria and fungi. The abundance of the bacterial phylum Acidobacteria was considerably different between the samples from the two regions. At genus level, Lactobacillus and Pichia were the predominating bacterial and fungal genera, respectively, while six bacterial genera significantly differed between the Kalmykia (enrichment of Aeromonas, Bacillus, Clostridium, Streptococcus, Vogesella) and Chita (enrichment of Lactococcus) samples. The results of principal coordinate analysis (PCoA) based on the bacterial or fungal composition of the Kalmykia and Chita samples revealed a different microbiota structure between the samples collected in these two locations. The redundancy analysis (RDA) identified 60 bacterial and 21 fungal OTUs as the key variables responsible for such microbiota structural difference. Our results suggest that structural differences existed in the microbiota of NFCM between Kalmykia and Chita. The difference in geographic environment may be an important factor influencing the microbial diversity of NFCM made by the Mongolians in Russia.

Changes in diversity, abundance, and structure of soil bacterial communities in Brazilian Savanna under different land use systems.

PubMed

Rampelotto, Pabulo Henrique; de Siqueira Ferreira, Adão; Barboza, Anthony Diego Muller; Roesch, Luiz Fernando Wurdig

2013-10-01

The Brazilian Savanna, also known as "Cerrado", is the richest and most diverse savanna in the world and has been ranked as one of the main hotspots of biodiversity. The Cerrado is a representative biome in Central Brazil and the second largest biome in species diversity of South America. Nevertheless, large areas of native vegetation have been converted to agricultural land including grain production, livestock, and forestry. In this view, understanding how land use affects microbial communities is fundamental for the sustainable management of agricultural ecosystems. The aim of this work was to analyze and compare the soil bacterial communities from the Brazilian Cerrado associated with different land use systems using high throughput pyrosequencing of 16S rRNA genes. Relevant differences were observed in the abundance and structure of bacterial communities in soils under different land use systems. On the other hand, the diversity of bacterial communities was not relevantly changed among the sites studied. Land use systems had also an important impact on specific bacterial groups in soil, which might change the soil function and the ecological processes. Acidobacteria, Proteobacteria, and Actinobacteria were the most abundant groups in the Brazilian Cerrado. These findings suggest that more important than analyzing the general diversity is to analyze the composition of the communities. Since soil type was the same among the sites, we might assume that land use was the main factor defining the abundance and structure of bacterial communities.

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

PubMed

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

2017-01-01

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

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

PubMed Central

JR, Luft; EH, Snell; GT, DeTitta

2011-01-01

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

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

PubMed

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.

Pyrosequencing of Plaque Microflora In Twin Children with Discordant Caries Phenotypes

PubMed Central

Zhang, Meng; Chen, Yongxing; Xie, Lingzhi; Li, Yuhong; Jiang, Han; Du, Minquan

2015-01-01

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. PMID:26524687

High-throughput determination of structural phase diagram and constituent phases using GRENDEL

NASA Astrophysics Data System (ADS)

Kusne, A. G.; Keller, D.; Anderson, A.; Zaban, A.; Takeuchi, I.

2015-11-01

Advances in high-throughput materials fabrication and characterization techniques have resulted in faster rates of data collection and rapidly growing volumes of experimental data. To convert this mass of information into actionable knowledge of material process-structure-property relationships requires high-throughput data analysis techniques. This work explores the use of the Graph-based endmember extraction and labeling (GRENDEL) algorithm as a high-throughput method for analyzing structural data from combinatorial libraries, specifically, to determine phase diagrams and constituent phases from both x-ray diffraction and Raman spectral data. The GRENDEL algorithm utilizes a set of physical constraints to optimize results and provides a framework by which additional physics-based constraints can be easily incorporated. GRENDEL also permits the integration of database data as shown by the use of critically evaluated data from the Inorganic Crystal Structure Database in the x-ray diffraction data analysis. Also the Sunburst radial tree map is demonstrated as a tool to visualize material structure-property relationships found through graph based analysis.

Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS)

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

Hura, Greg L.; Menon, Angeli L.; Hammel, Michal

2009-07-20

We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling of microliter volumes, temperature and anaerobic control, rapid data collection and data analysis, and couples structural analysis with automated archiving. We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline and found that 30 were multimeric structures in solution. SAXS analysis allowed us to distinguish aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and similar structures for 25 unknown structures, and determine envelopes formore » 41 proteins. We believe that high-throughput SAXS is an enabling technology that may change the way that structural genomics research is done.« less

Mining and Development of Novel SSR Markers Using Next Generation Sequencing (NGS) Data in Plants.

PubMed

Taheri, Sima; Lee Abdullah, Thohirah; Yusop, Mohd Rafii; Hanafi, Mohamed Musa; Sahebi, Mahbod; Azizi, Parisa; Shamshiri, Redmond Ramin

2018-02-13

Microsatellites, or simple sequence repeats (SSRs), are one of the most informative and multi-purpose genetic markers exploited in plant functional genomics. However, the discovery of SSRs and development using traditional methods are laborious, time-consuming, and costly. Recently, the availability of high-throughput sequencing technologies has enabled researchers to identify a substantial number of microsatellites at less cost and effort than traditional approaches. Illumina is a noteworthy transcriptome sequencing technology that is currently used in SSR marker development. Although 454 pyrosequencing datasets can be used for SSR development, this type of sequencing is no longer supported. This review aims to present an overview of the next generation sequencing, with a focus on the efficient use of de novo transcriptome sequencing (RNA-Seq) and related tools for mining and development of microsatellites in plants.

Unique hyper-thermal composting process in Kagoshima City forms distinct bacterial community structures.

PubMed

Tashiro, Yukihiro; Tabata, Hanae; Itahara, Asuka; Shimizu, Natsuki; Tashiro, Kosuke; Sakai, Kenji

2016-11-01

A unique compost, Satsuma soil, is produced from three types of wastewater sludge using hyper-thermal processes at temperatures much higher than that of general thermophilic processes in Kagoshima City, Japan. We analyzed the bacterial community structures of this hyper-thermal compost sample and other sludges and composts by a high-throughput barcoded pyrosequencing method targeting the 16S rRNA gene. In total, 621,076 reads were derived from 17 samples and filtered. Artificial sequences were deleted and the reads were clustered based on the operational taxonomic units (OTUs) at 97% similarity. Phylum-level analysis of the hyper-thermal compost revealed drastic changes of the sludge structures (each relative abundance) from Firmicutes (average 47.8%), Proteobacteria (average 22.3%), and Bacteroidetes (average 10.1%) to two main phyla including Firmicutes (73.6%) and Actinobacteria (25.0%) with less Proteobacteria (∼0.3%) and Bacteroidetes (∼0.1%). Furthermore, we determined the predominant species (each relative abundance) of the hyper-thermal compost including Firmicutes related to Staphylococcus cohnii (13.8%), Jeotgalicoccus coquinae (8.01%), and Staphylococcus lentus (5.96%), and Actinobacteria related to Corynebacterium stationis (6.41%), and found that these species were not predominant in wastewater sludge. In contrast, we did not observe any common structures among eight other composts produced, using the hyper-thermal composts as the inoculums, under thermophilic conditions from different materials. Principle coordinate analysis of the hyper-thermal compost indicated a large difference in bacterial community structures from material sludge and other composts. These results suggested that a distinct bacterial community structure was formed by hyper-thermal composting. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Preliminary characterization of the oral microbiota of Chinese adults with and without gingivitis

PubMed Central

2011-01-01

Background Microbial communities inhabiting human mouth are associated with oral health and disease. Previous studies have indicated the general prevalence of adult gingivitis in China to be high. The aim of this study was to characterize in depth the oral microbiota of Chinese adults with or without gingivitis, by defining the microbial phylogenetic diversity and community-structure using highly paralleled pyrosequencing. Methods Six non-smoking Chinese, three with and three without gingivitis (age range 21-39 years, 4 females and 2 males) were enrolled in the present cross-sectional study. Gingival parameters of inflammation and bleeding on probing were characterized by a clinician using the Mazza Gingival Index (MGI). Plaque (sampled separately from four different oral sites) and salivary samples were obtained from each subject. Sequences and relative abundance of the bacterial 16 S rDNA PCR-amplicons were determined via pyrosequencing that produced 400 bp-long reads. The sequence data were analyzed via a computational pipeline customized for human oral microbiome analyses. Furthermore, the relative abundances of selected microbial groups were validated using quantitative PCR. Results The oral microbiomes from gingivitis and healthy subjects could be distinguished based on the distinct community structures of plaque microbiomes, but not the salivary microbiomes. Contributions of community members to community structure divergence were statistically accessed at the phylum, genus and species-like levels. Eight predominant taxa were found associated with gingivitis: TM7, Leptotrichia, Selenomonas, Streptococcus, Veillonella, Prevotella, Lautropia, and Haemophilus. Furthermore, 98 species-level OTUs were identified to be gingivitis-associated, which provided microbial features of gingivitis at a species resolution. Finally, for the two selected genera Streptococcus and Fusobacterium, Real-Time PCR based quantification of relative bacterial abundance validated the pyrosequencing-based results. Conclusions This methods study suggests that oral samples from this patient population of gingivitis can be characterized via plaque microbiome by pyrosequencing the 16 S rDNA genes. Further studies that characterize serial samples from subjects (longitudinal study design) with a larger population size may provide insight into the temporal and ecological features of oral microbial communities in clinically-defined states of gingivitis. PMID:22152152

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

PubMed

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.

Dysbiosis of oral buccal mucosa microbiota in patients with oral lichen planus.

PubMed

He, Y; Gong, D; Shi, C; Shao, F; Shi, J; Fei, J

2017-07-01

The bacterial community structure of buccal mucosa in patients with oral lichen planus was evaluated and compared with healthy control. Buccal scraping samples have been taken on 43 oral lichen planus patients (21 erosive and 22 non-erosive) and 21 mucosal healthy volunteers. The V3 hypervariable 16S rDNA region was amplified and sequenced by high-throughput 454 pyrosequencing. 94.26% of the total buccal bacteria were classified into 15 abundant genera. Eight of these abundant genera could be detected in all cases, namely Streptococcus, Prevotella, Haemophilu, Neisseria, Fusobacterium, Leptotrichia, Veillonella and Actinomyces. Four abundant bacteria showed significantly different prevalence at the genus level: Streptococcus was more abundant (P < 0.01) in healthy control group, while Fusobacterium (P < 0.01), Leptotrichia (P < 0.001) and Lautropia (P < 0.001) showed higher abundance in OLP group. Few differences can be found between erosive and non-erosive OLP. In general, 19 'core' OTUs at three levels were defined to be OLP specific, comparing with healthy control group. These results suggest that OLP is associated with dysbiosis of the oral microbiome. Further studies should be taken to elucidate the inner relationship between these observed changes and OLP development. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Lentinula edodes-Derived Polysaccharide Alters the Spatial Structure of Gut Microbiota in Mice

PubMed Central

Xu, Xiaofei; Zhang, Xuewu

2015-01-01

Lentinula edodes-derived polysaccharides possess many therapeutic characteristics, including anti-tumor and immuno-modulation. The gut microbes play a critical role in modulation of immune function. However, the impact of Lentinula edodes-derived polysaccharides on the gut microbes have not yet been explored. In this study, high-throughput pyrosequencing technique was employed to investigate the effects of a new heteropolysaccharide L2 from Lentinula edodes on microbiota diversity and composition of small intestine, cecum, colon and distal end of colon (feces) in mice. The results demonstrated that along mouse intestine the microbiota exhibit distinctly different space distribution. L2 treatment reduced the diversity and evenness of gut microbiota along the intestine, especially in the cecum and colon. In the fecal microbial communities, the decrease of Bacteroidetes by significantly increasing Proteobacteria were observed, which were characterized by the increased Helicobacteraceae and reduced S24-7 at family level. Some OTUs, corresponding to Bacteroides acidifaciens, Alistipes and Helicobacter suncus, were found to be significantly increased in L2 treated-mice. In particular, 4 phyla Chloroflexi, Gemmatimonadetes, Nitrospirae and Planctomycetes are exclusively present in L2-treated mice. This is helpful for further demonstrating healthy action mechanism of Lentinula edodes-derived polysaccharide L2. PMID:25608087

Seasonality Affects the Diversity and Composition of Bacterioplankton Communities in Dongjiang River, a Drinking Water Source of Hong Kong.

PubMed

Sun, Wei; Xia, Chunyu; Xu, Meiying; Guo, Jun; Sun, Guoping

2017-01-01

Water quality ranks the most vital criterion for rivers serving as drinking water sources, which periodically changes over seasons. Such fluctuation is believed associated with the state shifts of bacterial community within. To date, seasonality effects on bacterioplankton community patterns in large rivers serving as drinking water sources however, are still poorly understood. Here we investigated the intra-annual bacterial community structure in the Dongjiang River, a drinking water source of Hong Kong, using high-throughput pyrosequencing in concert with geochemical property measurements during dry, and wet seasons. Our results showed that Proteobacteria, Actinobacteria , and Bacteroidetes were the dominant phyla of bacterioplankton communities, which varied in composition, and distribution from dry to wet seasons, and exhibited profound seasonal changes. Actinobacteria, Bacteroidetes , and Cyanobacteria seemed to be more associated with seasonality that the relative abundances of Actinobacteria , and Bacteroidetes were significantly higher in the dry season than those in the wet season ( p < 0.01), while the relative abundance of Cyanobacteria was about 10-fold higher in the wet season than in the dry season. Temperature and [Formula: see text]-N concentration represented key contributing factors to the observed seasonal variations. These findings help understand the roles of various bacterioplankton and their interactions with the biogeochemical processes in the river ecosystem.

Characterization of the microbial community structure and nitrosamine-reducing isolates in drinking water biofilters.

PubMed

Wang, Wanfeng; Guo, Yanling; Yang, Qingxiang; Huang, Yao; Zhu, Chunyou; Fan, Jing; Pan, Feng

2015-07-15

Two biofilters were constructed using biological activated carbon (BAC) and nitrosamine-containing water from two drinking water treatment plants. The microbiome of each biofilter was characterized by 454 high-throughput pyrosequencing, and one nitrosamine-reducing bacterium was isolated. The results showed that nitrosamines changed the relative abundance at both the phylum and class levels, and the new genera were observed in the microbial communities of the two BAC filters after cultivation. As such, the genus Rhodococcus, which includes many nitrosamine-reducing strains reported in previous studies, was only detected in the BAC2 filter after cultivation. These findings indicate that nitrosamines can significantly affect the genus level in the microbial communities. Furthermore, the isolated bacterial culture Rhodococcus cercidiphylli A41 AS-1 exhibited the ability to reduce five nitrosamines (N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosodi-n-propylamine, N-nitrosopyrrolidine, and N-nitrosodi-n-butylamine) with removal ratios that ranged from 38.1% to 85.4%. The isolate exhibited a better biodegradation ability with nitrosamine as the carbon source when compared with nitrosamine as the nitrogen source. This study increases our understanding of the microbial community in drinking water biofilters with trace quantities of nitrosamines, and provides information on the metabolism of nitrosamine-reducing bacteria. Copyright © 2015. Published by Elsevier B.V.

Microbial Diversity in a Permanently Cold and Alkaline Environment in Greenland

PubMed Central

Glaring, Mikkel A.; Vester, Jan K.; Lylloff, Jeanette E.; Abu Al-Soud, Waleed; Sørensen, Søren J.; Stougaard, Peter

2015-01-01

The submarine ikaite columns located in the Ikka Fjord in Southern Greenland represent a unique, permanently cold (less than 6°C) and alkaline (above pH 10) environment and are home to a microbial community adapted to these extreme conditions. The bacterial and archaeal community inhabiting the ikaite columns and surrounding fjord was characterised by high-throughput pyrosequencing of 16S rRNA genes. Analysis of the ikaite community structure revealed the presence of a diverse bacterial community, both in the column interior and at the surface, and very few archaea. A clear difference in overall taxonomic composition was observed between column interior and surface. Whereas the surface, and in particular newly formed ikaite material, was primarily dominated by Cyanobacteria and phototrophic Proteobacteria, the column interior was dominated by Proteobacteria and putative anaerobic representatives of the Firmicutes and Bacteroidetes. The results suggest a stratification of the ikaite columns similar to that of classical soda lakes, with a light-exposed surface inhabited by primary producers and an anoxic subsurface. This was further supported by identification of major taxonomic groups with close relatives in soda lake environments, including members of the genera Rhodobaca, Dethiobacter, Thioalkalivibrio and Tindallia, as well as very abundant groups related to uncharacterised environmental sequences originally isolated from Mono Lake in California. PMID:25915866

Microbial diversity in a permanently cold and alkaline environment in Greenland.

PubMed

Glaring, Mikkel A; Vester, Jan K; Lylloff, Jeanette E; Al-Soud, Waleed Abu; Sørensen, Søren J; Stougaard, Peter

2015-01-01

The submarine ikaite columns located in the Ikka Fjord in Southern Greenland represent a unique, permanently cold (less than 6°C) and alkaline (above pH 10) environment and are home to a microbial community adapted to these extreme conditions. The bacterial and archaeal community inhabiting the ikaite columns and surrounding fjord was characterised by high-throughput pyrosequencing of 16S rRNA genes. Analysis of the ikaite community structure revealed the presence of a diverse bacterial community, both in the column interior and at the surface, and very few archaea. A clear difference in overall taxonomic composition was observed between column interior and surface. Whereas the surface, and in particular newly formed ikaite material, was primarily dominated by Cyanobacteria and phototrophic Proteobacteria, the column interior was dominated by Proteobacteria and putative anaerobic representatives of the Firmicutes and Bacteroidetes. The results suggest a stratification of the ikaite columns similar to that of classical soda lakes, with a light-exposed surface inhabited by primary producers and an anoxic subsurface. This was further supported by identification of major taxonomic groups with close relatives in soda lake environments, including members of the genera Rhodobaca, Dethiobacter, Thioalkalivibrio and Tindallia, as well as very abundant groups related to uncharacterised environmental sequences originally isolated from Mono Lake in California.

A morphogenetic survey on ciliate plankton from a mountain lake pinpoints the necessity of lineage-specific barcode markers in microbial ecology.

PubMed

Stoeck, Thorsten; Breiner, Hans-Werner; Filker, Sabine; Ostermaier, Veronika; Kammerlander, Barbara; Sonntag, Bettina

2014-02-01

Analyses of high-throughput environmental sequencing data have become the 'gold-standard' to address fundamental questions of microbial diversity, ecology and biogeography. Findings that emerged from sequencing are, e.g. the discovery of the extensive 'rare microbial biosphere' and its potential function as a seed-bank. Even though applied since several years, results from high-throughput environmental sequencing have hardly been validated. We assessed how well pyrosequenced amplicons [the hypervariable eukaryotic V4 region of the small subunit ribosomal RNA (SSU rRNA) gene] reflected morphotype ciliate plankton. Moreover, we assessed if amplicon sequencing had the potential to detect the annual ciliate plankton stock. In both cases, we identified significant quantitative and qualitative differences. Our study makes evident that taxon abundance distributions inferred from amplicon data are highly biased and do not mirror actual morphotype abundances at all. Potential reasons included cell losses after fixation, cryptic morphotypes, resting stages, insufficient sequence data availability of morphologically described species and the unsatisfying resolution of the V4 SSU rRNA fragment for accurate taxonomic assignments. The latter two underline the necessity of barcoding initiatives for eukaryotic microbes to better and fully exploit environmental amplicon data sets, which then will also allow studying the potential of seed-bank taxa as a buffer for environmental changes. © 2013 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

A high-throughput exploration of magnetic materials by using structure predicting methods

NASA Astrophysics Data System (ADS)

Arapan, S.; Nieves, P.; Cuesta-López, S.

2018-02-01

We study the capability of a structure predicting method based on genetic/evolutionary algorithm for a high-throughput exploration of magnetic materials. We use the USPEX and VASP codes to predict stable and generate low-energy meta-stable structures for a set of representative magnetic structures comprising intermetallic alloys, oxides, interstitial compounds, and systems containing rare-earths elements, and for both types of ferromagnetic and antiferromagnetic ordering. We have modified the interface between USPEX and VASP codes to improve the performance of structural optimization as well as to perform calculations in a high-throughput manner. We show that exploring the structure phase space with a structure predicting technique reveals large sets of low-energy metastable structures, which not only improve currently exiting databases, but also may provide understanding and solutions to stabilize and synthesize magnetic materials suitable for permanent magnet applications.

Microbial reduction and precipitation of vanadium (V) in groundwater by immobilized mixed anaerobic culture.

PubMed

Zhang, Baogang; Hao, Liting; Tian, Caixing; Yuan, Songhu; Feng, Chuanping; Ni, Jinren; Borthwick, Alistair G L

2015-09-01

Vanadium is an important contaminant impacted by natural and industrial activities. Vanadium (V) reduction efficiency as high as 87.0% was achieved by employing immobilized mixed anaerobic sludge as inoculated seed within 12h operation, while V(IV) was the main reduction product which precipitated instantly. Increasing initial V(V) concentration resulted in the decrease of V(V) removal efficiency, while this index increased first and then decreased with the increase of initial COD concentration, pH and conductivity. High-throughput 16S rRNA gene pyrosequencing analysis indicated the decreased microbial diversity. V(V) reduction was realized through dissimilatory reduction process by significantly enhanced Lactococcus and Enterobacter with oxidation of lactic and acetic acids from fermentative microorganisms such as the enriched Paludibacter and the newly appeared Acetobacterium, Oscillibacter. This study is helpful to detect new functional species for V(V) reduction and constitutes a step ahead in developing in situ bioremediations of vanadium contamination. Copyright © 2015 Elsevier Ltd. All rights reserved.

Nanoliter reactors improve multiple displacement amplification of genomes from single cells.

PubMed

Marcy, Yann; Ishoey, Thomas; Lasken, Roger S; Stockwell, Timothy B; Walenz, Brian P; Halpern, Aaron L; Beeson, Karen Y; Goldberg, Susanne M D; Quake, Stephen R

2007-09-01

Since only a small fraction of environmental bacteria are amenable to laboratory culture, there is great interest in genomic sequencing directly from single cells. Sufficient DNA for sequencing can be obtained from one cell by the Multiple Displacement Amplification (MDA) method, thereby eliminating the need to develop culture methods. Here we used a microfluidic device to isolate individual Escherichia coli and amplify genomic DNA by MDA in 60-nl reactions. Our results confirm a report that reduced MDA reaction volume lowers nonspecific synthesis that can result from contaminant DNA templates and unfavourable interaction between primers. The quality of the genome amplification was assessed by qPCR and compared favourably to single-cell amplifications performed in standard 50-microl volumes. Amplification bias was greatly reduced in nanoliter volumes, thereby providing a more even representation of all sequences. Single-cell amplicons from both microliter and nanoliter volumes provided high-quality sequence data by high-throughput pyrosequencing, thereby demonstrating a straightforward route to sequencing genomes from single cells.

Lignases and aldo-keto reductases for conversion of lignin-containing materials to fermentable products

DOEpatents

Scharf, Michael; Sethi, Amit

2016-09-13

Termites have specialized digestive systems that overcome the lignin barrier in wood to release fermentable simple sugars. Using the termite Reticulitermes flavipes and its gut symbionts, high-throughput titanium pyrosequencing and proteomics approaches experimentally compared the effects of lignin-containing diets on host-symbiont digestome composition. Proteomic investigations and functional digestive studies with recombinant lignocellulases conducted in parallel provided strong evidence of congruence at the transcription and translational levels and provide enzymatic strategies for overcoming recalcitrant lignin barriers in biofuel feedstocks. Briefly described, therefore, the disclosure provides a system for generating a fermentable product from a lignified plant material, the system comprising a cooperating series of at least two catalytically active polypeptides, where said catalytically active polypeptides are selected from the group consisting of: cellulase Cell-1, .beta.-glu cellulase, an aldo-keto-reductase, a catalase, a laccase, and an endo-xylanase.

Short communication: Evaluation of the microbiota of kefir samples using metagenetic analysis targeting the 16S and 26S ribosomal DNA fragments.

PubMed

Korsak, N; Taminiau, B; Leclercq, M; Nezer, C; Crevecoeur, S; Ferauche, C; Detry, E; Delcenserie, V; Daube, G

2015-06-01

Milk kefir is produced by fermenting milk in the presence of kefir grains. This beverage has several benefits for human health. The aim of this experiment was to analyze 5 kefir grains (and their products) using a targeted metagenetic approach. Of the 5 kefir grains analyzed, 1 was purchased in a supermarket, 2 were provided by the Ministry of Agriculture (Namur, Belgium), and 2 were provided by individuals. The metagenetic approach targeted the V1-V3 fragment of the 16S ribosomal (r)DNA for the grains and the resulting beverages at 2 levels of grain incorporation (5 and 10%) to identify the bacterial species population. In contrast, the 26S rDNA pyrosequencing was performed only on kefir grains with the aim of assessing the yeast populations. In parallel, pH measurements were performed on the kefir obtained from the kefir grains using 2 incorporation rates. Regarding the bacterial population, 16S pyrosequencing revealed the presence of 20 main bacterial species, with a dominance of the following: Lactobacillus kefiranofaciens, Lactococcus lactis ssp. cremoris, Gluconobacter frateurii, Lactobacillus kefiri, Acetobacter orientalis, and Acetobacter lovaniensis. An important difference was noticed between the kefir samples: kefir grain purchased from a supermarket (sample E) harbored a much higher proportion of several operational taxonomic units of Lactococcus lactis and Leuconostoc mesenteroides. This sample of grain was macroscopically different from the others in terms of size, apparent cohesion of the grains, structure, and texture, probably associated with a lower level of Lactobacillus kefiranofaciens. The kefir (at an incorporation rate of 5%) produced from this sample of grain was characterized by a lower pH value (4.5) than the others. The other 4 samples of kefir (5%) had pH values above 5. Comparing the kefir grain and the kefir, an increase in the population of Gluconobacter in grain sample B was observed. This was also the case for Acetobacter orientalis in sample D. In relation to 26S pyrosequencing, our study revealed the presence of 3 main yeast species: Naumovozyma spp., Kluyveromyces marxianus, and Kazachastania khefir. For Naumovozyma, further studies are needed to assess the isolation of new species. In conclusion, this study has proved that it is possible to establish the patterns of bacterial and yeast composition of kefir and kefir grain. This was only achieved with the use of high-throughput sequencing techniques. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

A facile and cost-effective TEM grid approach to design gold nano-structured substrates for high throughput plasmonic sensitive detection of biomolecules.

PubMed

Jia, Kun; Bijeon, Jean Louis; Adam, Pierre Michel; Ionescu, Rodica Elena

2013-02-21

A commercial TEM grid was used as a mask for the creation of extremely well-organized gold micro-/nano-structures on a glass substrate via a high temperature annealing process at 500 °C. The structured substrate was (bio)functionalized and used for the high throughput LSPR immunosensing of different concentrations of a model protein named bovine serum albumin.

Fecal microbiota of lambs fed purple prairie clover (Dalea purpurea Vent.) and alfalfa (Medicago sativa).

PubMed

Huang, Qianqian; Holman, Devin B; Alexander, Trevor; Hu, Tianming; Jin, Long; Xu, Zhongjun; McAllister, Tim A; Acharya, Surya; Zhao, Guoqi; Wang, Yuxi

2018-01-01

The present study assessed the effect of purple prairie clover (PPC) and PPC condensed tannins (CT) on the fecal microbiota of lambs using high-throughput 16S rRNA gene pyrosequencing. A total of 18 individual lambs were randomly divided into three groups and fed either green chop alfalfa (Alf), a 40:60 (DM basis; Mix) mixture of Alf and PPC, or Mix supplemented with polyethylene glycol (Mix-P) for 18 days. Fecal samples were collected on days 13 through 18 using digital rectal retrieval. The DNA of fecal samples was extracted and the microbial 16S rRNA gene amplicons were sequenced using 454 pyrosequencing. Regardless of diet, the bacterial community was dominated by Firmicutes and Bacteroidetes with many sequences unclassified at the genus level. Forage type and CT had no effect on the fecal microbial composition at the phylum level or on α-diversity. Compared to the Alf diet, the Mix diet reduced the relative abundance of Akkermansia (P = 0.03) and Asteroleplasma (P = 0.05). Fecal microbial populations in Alf and Mix-P clustered separately from each other when assessed using unweighted UniFrac (P < 0.05). These results indicate that PPC CT up to 36 g/kg DM in the diet had no major effect on fecal microbial flora at the phyla level and exerted only minor effects on the genera composition of fecal microbiota in lambs.

A Pyrosequencing-Based Assay for the Rapid Detection of the 22q11.2 Deletion in DNA from Buccal and Dried Blood Spot Samples

PubMed Central

Koontz, Deborah; Baecher, Kirsten; Kobrynski, Lisa; Nikolova, Stanimila; Gallagher, Margaret

2015-01-01

The 22q11.2 deletion syndrome is one of the most common deletion syndromes in newborns. Some affected newborns may be diagnosed shortly after birth because of the presence of heart defects, palatal defects, or severe immune deficiencies. However, diagnosis is often delayed in patients presenting with other associated conditions that would benefit from early recognition and treatment, such as speech delays, learning difficulties, and schizophrenia. Fluorescence in situ hybridization (FISH) is the gold standard for deletion detection, but it is costly and time consuming and requires a whole blood specimen. Our goal was to develop a suitable assay for population-based screening of easily collectible specimens, such as buccal swabs and dried blood spots (DBS). We designed a pyrosequencing assay and validated it using DNA from FISH–confirmed 22q11 deletion syndrome patients and normal controls. We tested DBS from nine patients and paired buccal cell and venous blood specimens from 20 patients. Results were 100% concordant with FISH assay results. DNA samples from normal controls (n = 180 cell lines, n = 15 DBS, and n = 88 buccal specimens) were negative for the deletion. Limiting dilution experiments demonstrated that accurate results could be obtained from as little as 1 ng of DNA. This method represents a reliable and low-cost alternative for detection of the common 22q11.2 microdeletions and can be adapted to high-throughput population screening. PMID:24973633

Diversity and homogeneity of oral microbiota in healthy Korean pre-school children using pyrosequencing.

PubMed

Lee, Soo Eon; Nam, Ok Hyung; Lee, Hyo-Seol; Choi, Sung Chul

2016-07-01

Objectives The purpose of this study was designed to identify the oral microbiota in healthy Korean pre-school children using pyrosequencing. Materials and methods Dental plaque samples were obtained form 10 caries-free pre-school children. The samples were analysed using pyrosequencing. Results The pyrosequencing analysis revealed that, at the phylum level, Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria and Fusobacteria showed high abundance. Also, predominant genera were identified as core microbiome, such as Streptococcus, Neisseria, Capnocytophaga, Haemophilus and Veilonella. Conclusions The diversity and homogeneity was shown in the dental plaque microbiota in healthy Korean pre-school children.

High performance hybrid magnetic structure for biotechnology applications

DOEpatents

Humphries, David E; Pollard, Martin J; Elkin, Christopher J

2005-10-11

The present disclosure provides a high performance hybrid magnetic structure made from a combination of permanent magnets and ferromagnetic pole materials which are assembled in a predetermined array. The hybrid magnetic structure provides means for separation and other biotechnology applications involving holding, manipulation, or separation of magnetizable molecular structures and targets. Also disclosed are: a method of assembling the hybrid magnetic plates, a high throughput protocol featuring the hybrid magnetic structure, and other embodiments of the ferromagnetic pole shape, attachment and adapter interfaces for adapting the use of the hybrid magnetic structure for use with liquid handling and other robots for use in high throughput processes.

High performance hybrid magnetic structure for biotechnology applications

DOEpatents

Humphries, David E.; Pollard, Martin J.; Elkin, Christopher J.

2006-12-12

The present disclosure provides a high performance hybrid magnetic structure made from a combination of permanent magnets and ferromagnetic pole materials which are assembled in a predetermined array. The hybrid magnetic structure provides for separation and other biotechnology applications involving holding, manipulation, or separation of magnetic or magnetizable molecular structures and targets. Also disclosed are: a method of assembling the hybrid magnetic plates, a high throughput protocol featuring the hybrid magnetic structure, and other embodiments of the ferromagnetic pole shape, attachment and adapter interfaces for adapting the use of the hybrid magnetic structure for use with liquid handling and other robots for use in high throughput processes.

A 0.13-µm implementation of 5 Gb/s and 3-mW folded parallel architecture for AES algorithm

NASA Astrophysics Data System (ADS)

Rahimunnisa, K.; Karthigaikumar, P.; Kirubavathy, J.; Jayakumar, J.; Kumar, S. Suresh

2014-02-01

A new architecture for encrypting and decrypting the confidential data using Advanced Encryption Standard algorithm is presented in this article. This structure combines the folded structure with parallel architecture to increase the throughput. The whole architecture achieved high throughput with less power. The proposed architecture is implemented in 0.13-µm Complementary metal-oxide-semiconductor (CMOS) technology. The proposed structure is compared with different existing structures, and from the result it is proved that the proposed structure gives higher throughput and less power compared to existing works.

Comparison of Bacterial Community Composition of Primary and Persistent Endodontic Infections Using Pyrosequencing.

PubMed

Tzanetakis, Giorgos N; Azcarate-Peril, M Andrea; Zachaki, Sophia; Panopoulos, Panos; Kontakiotis, Evangelos G; Madianos, Phoebus N; Divaris, Kimon

2015-08-01

Elucidating the microbial ecology of endodontic infections (EIs) is a necessary step in developing effective intracanal antimicrobials. The aim of the present study was to investigate the bacterial composition of symptomatic and asymptomatic primary and persistent infections in a Greek population using high-throughput sequencing methods. 16S amplicon pyrosequencing of 48 root canal bacterial samples was conducted, and sequencing data were analyzed using an oral microbiome-specific and a generic (Greengenes) database. Bacterial abundance and diversity were examined by EI type (primary or persistent), and statistical analysis was performed by using non-parametric and parametric tests accounting for clustered data. Bacteroidetes was the most abundant phylum in both infection groups. Significant, albeit weak associations of bacterial diversity were found, as measured by UniFrac distances with infection type (analyses of similarity, R = 0.087, P = .005) and symptoms (analyses of similarity, R = 0.055, P = .047). Persistent infections were significantly enriched for Proteobacteria and Tenericutes compared with primary ones; at the genus level, significant differences were noted for 14 taxa, including increased enrichment of persistent infections for Lactobacillus, Streptococcus, and Sphingomonas. More but less abundant phyla were identified using the Greengenes database; among those, Cyanobacteria (0.018%) and Acidobacteria (0.007%) were significantly enriched among persistent infections. Persistent infections showed higher phylogenetic diversity (PD) (asymptomatic: PD = 9.2, standard error [SE] = 1.3; symptomatic: PD = 8.2, SE = 0.7) compared with primary infections (asymptomatic: PD = 5.9, SE = 0.8; symptomatic: PD = 7.4, SE = 1.0). The present study revealed a high bacterial diversity of EI and suggests that persistent infections may have more diverse bacterial communities than primary infections. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

A pyrosequencing-based metagenomic study of methane-producing microbial community in solid-state biogas reactor

PubMed Central

2013-01-01

Background A solid-state anaerobic digestion method is used to produce biogas from various solid wastes in China but the efficiency of methane production requires constant improvement. The diversity and abundance of relevant microorganisms play important roles in methanogenesis of biomass. The next-generation high-throughput pyrosequencing platform (Roche/454 GS FLX Titanium) provides a powerful tool for the discovery of novel microbes within the biogas-generating microbial communities. Results To improve the power of our metagenomic analysis, we first evaluated five different protocols for extracting total DNA from biogas-producing mesophilic solid-state fermentation materials and then chose two high-quality protocols for a full-scale analysis. The characterization of both sequencing reads and assembled contigs revealed that the most prevalent microbes of the fermentation materials are derived from Clostridiales (Firmicutes), which contribute to degrading both protein and cellulose. Other important bacterial species for decomposing fat and carbohydrate are Bacilli, Gammaproteobacteria, and Bacteroidetes (belonging to Firmicutes, Proteobacteria, and Bacteroidetes, respectively). The dominant bacterial species are from six genera: Clostridium, Aminobacterium, Psychrobacter, Anaerococcus, Syntrophomonas, and Bacteroides. Among them, abundant Psychrobacter species, which produce low temperature-adaptive lipases, and Anaerococcus species, which have weak fermentation capabilities, were identified for the first time in biogas fermentation. Archaea, represented by genera Methanosarcina, Methanosaeta and Methanoculleus of Euryarchaeota, constitute only a small fraction of the entire microbial community. The most abundant archaeal species include Methanosarcina barkeri fusaro, Methanoculleus marisnigri JR1, and Methanosaeta theromphila, and all are involved in both acetotrophic and hydrogenotrophic methanogenesis. Conclusions The identification of new bacterial genera and species involved in biogas production provides insights into novel designs of solid-state fermentation under mesophilic or low-temperature conditions. PMID:23320936

X-ray transparent microfluidic chips for high-throughput screening and optimization of in meso membrane protein crystallization

PubMed Central

Schieferstein, Jeremy M.; Pawate, Ashtamurthy S.; Wan, Frank; Sheraden, Paige N.; Broecker, Jana; Ernst, Oliver P.; Gennis, Robert B.

2017-01-01

Elucidating and clarifying the function of membrane proteins ultimately requires atomic resolution structures as determined most commonly by X-ray crystallography. Many high impact membrane protein structures have resulted from advanced techniques such as in meso crystallization that present technical difficulties for the set-up and scale-out of high-throughput crystallization experiments. In prior work, we designed a novel, low-throughput X-ray transparent microfluidic device that automated the mixing of protein and lipid by diffusion for in meso crystallization trials. Here, we report X-ray transparent microfluidic devices for high-throughput crystallization screening and optimization that overcome the limitations of scale and demonstrate their application to the crystallization of several membrane proteins. Two complementary chips are presented: (1) a high-throughput screening chip to test 192 crystallization conditions in parallel using as little as 8 nl of membrane protein per well and (2) a crystallization optimization chip to rapidly optimize preliminary crystallization hits through fine-gradient re-screening. We screened three membrane proteins for new in meso crystallization conditions, identifying several preliminary hits that we tested for X-ray diffraction quality. Further, we identified and optimized the crystallization condition for a photosynthetic reaction center mutant and solved its structure to a resolution of 3.5 Å. PMID:28469762

Characterization of the relative importance of human- and infrastructure-associated bacteria in grey water: a case study.

PubMed

Keely, S P; Brinkman, N E; Zimmerman, B D; Wendell, D; Ekeren, K M; De Long, S K; Sharvelle, S; Garland, J L

2015-07-01

Development of efficacious grey water (GW) treatment systems would benefit from detailed knowledge of the bacterial composition of GW. Thus, the aim of this study was to characterize the bacterial composition from (i) various points throughout a GW recycling system that collects shower and sink handwash (SH) water into an equalization tank (ET) prior to treatment and (ii) laundry (LA) water effluent of a commercial-scale washer. Bacterial composition was analysed by high-throughput pyrosequencing of the 16S rRNA gene. LA was dominated by skin-associated bacteria, with Corynebacterium, Staphylococcus, Micrococcus, Propionibacterium and Lactobacillus collectively accounting for nearly 50% of the total sequences. SH contained a more evenly distributed community than LA, with some overlap (e.g. Propionibacterium), but also contained distinct genera common to wastewater infrastructure (e.g. Zoogloea). The ET contained many of these same wastewater infrastructure-associated bacteria, but was dominated by genera adapted for anaerobic conditions. The data indicate that a relatively consistent set of skin-associated genera are the dominant human-associated bacteria in GW, but infrastructure-associated bacteria from the GW collection system and ET used for transient storage will be the most common bacteria entering GW treatment and reuse systems. This study is the first to use high-throughput sequencing to identify the bacterial composition of various GW sources. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

Characterization of the fecal microbiota using high-throughput sequencing reveals a stable microbial community during storage.

PubMed

Carroll, Ian M; Ringel-Kulka, Tamar; Siddle, Jennica P; Klaenhammer, Todd R; Ringel, Yehuda

2012-01-01

The handling and treatment of biological samples is critical when characterizing the composition of the intestinal microbiota between different ecological niches or diseases. Specifically, exposure of fecal samples to room temperature or long term storage in deep freezing conditions may alter the composition of the microbiota. Thus, we stored fecal samples at room temperature and monitored the stability of the microbiota over twenty four hours. We also investigated the stability of the microbiota in fecal samples during a six month storage period at -80°C. As the stability of the fecal microbiota may be affected by intestinal disease, we analyzed two healthy controls and two patients with irritable bowel syndrome (IBS). We used high-throughput pyrosequencing of the 16S rRNA gene to characterize the microbiota in fecal samples stored at room temperature or -80°C at six and seven time points, respectively. The composition of microbial communities in IBS patients and healthy controls were determined and compared using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline. The composition of the microbiota in fecal samples stored for different lengths of time at room temperature or -80°C clustered strongly based on the host each sample originated from. Our data demonstrates that fecal samples exposed to room or deep freezing temperatures for up to twenty four hours and six months, respectively, exhibit a microbial composition and diversity that shares more identity with its host of origin than any other sample.

Multiplex SNaPshot-a new simple and efficient CYP2D6 and ADRB1 genotyping method.

PubMed

Ben, Songtao; Cooper-DeHoff, Rhonda M; Flaten, Hanna K; Evero, Oghenero; Ferrara, Tracey M; Spritz, Richard A; Monte, Andrew A

2016-04-23

Reliable, inexpensive, high-throughput genotyping methods are required for clinical trials. Traditional assays require numerous enzyme digestions or are too expensive for large sample volumes. Our objective was to develop an inexpensive, efficient, and reliable assay for CYP2D6 and ADRB1 accounting for numerous polymorphisms including gene duplications. We utilized the multiplex SNaPshot® custom genotype method to genotype CYP2D6 and ADRB1. We compared the method to reference standards genotyped using the Taqman Copy Number Variant Assay followed by pyrosequencing quantification and determined assigned genotype concordance. We genotyped 119 subjects. Seven (5.9 %) were found to be CYP2D6 poor metabolizers (PMs), 18 (15.1 %) intermediate metabolizers (IMs), 89 (74.8 %) extensive metabolizers (EMs), and 5 (4.2 %) ultra-rapid metabolizers (UMs). We genotyped two variants in the β1-adrenoreceptor, rs1801253 (Gly389Arg) and rs1801252 (Ser49Gly). The Gly389Arg genotype is Gly/Gly 18 (15.1 %), Gly/Arg 58 (48.7 %), and Arg/Arg 43 (36.1 %). The Ser49Gly genotype is Ser/Ser 82 (68.9 %), Ser/Gly 32 (26.9), and Gly/Gly 5 (4.2 %). The multiplex SNaPshot method was concordant with genotypes in reference samples. The multiplex SNaPshot method allows for specific and accurate detection of CYP2D6 genotypes and ADRB1 genotypes and haplotypes. This platform is simple and efficient and suited for high throughput.

Differential resistance of drinking water bacterial populations to monochloramine disinfection.

PubMed

Chiao, Tzu-Hsin; Clancy, Tara M; Pinto, Ameet; Xi, Chuanwu; Raskin, Lutgarde

2014-04-01

The impact of monochloramine disinfection on the complex bacterial community structure in drinking water systems was investigated using culture-dependent and culture-independent methods. Changes in viable bacterial diversity were monitored using culture-independent methods that distinguish between live and dead cells based on membrane integrity, providing a highly conservative measure of viability. Samples were collected from lab-scale and full-scale drinking water filters exposed to monochloramine for a range of contact times. Culture-independent detection of live cells was based on propidium monoazide (PMA) treatment to selectively remove DNA from membrane-compromised cells. Quantitative PCR (qPCR) and pyrosequencing of 16S rRNA genes was used to quantify the DNA of live bacteria and characterize the bacterial communities, respectively. The inactivation rate determined by the culture-independent PMA-qPCR method (1.5-log removal at 664 mg·min/L) was lower than the inactivation rate measured by the culture-based methods (4-log removal at 66 mg·min/L). Moreover, drastic changes in the live bacterial community structure were detected during monochloramine disinfection using PMA-pyrosequencing, while the community structure appeared to remain stable when pyrosequencing was performed on samples that were not subject to PMA treatment. Genera that increased in relative abundance during monochloramine treatment include Legionella, Escherichia, and Geobacter in the lab-scale system and Mycobacterium, Sphingomonas, and Coxiella in the full-scale system. These results demonstrate that bacterial populations in drinking water exhibit differential resistance to monochloramine, and that the disinfection process selects for resistant bacterial populations.

New FeFe-hydrogenase genes identified in a metagenomic fosmid library from a municipal wastewater treatment plant as revealed by high-throughput sequencing.

PubMed

Tomazetto, Geizecler; Wibberg, Daniel; Schlüter, Andreas; Oliveira, Valéria M

2015-01-01

A fosmid metagenomic library was constructed with total community DNA obtained from a municipal wastewater treatment plant (MWWTP), with the aim of identifying new FeFe-hydrogenase genes encoding the enzymes most important for hydrogen metabolism. The dataset generated by pyrosequencing of a fosmid library was mined to identify environmental gene tags (EGTs) assigned to FeFe-hydrogenase. The majority of EGTs representing FeFe-hydrogenase genes were affiliated with the class Clostridia, suggesting that this group is the main hydrogen producer in the MWWTP analyzed. Based on assembled sequences, three FeFe-hydrogenase genes were predicted based on detection of the L2 motif (MPCxxKxxE) in the encoded gene product, confirming true FeFe-hydrogenase sequences. These sequences were used to design specific primers to detect fosmids encoding FeFe-hydrogenase genes predicted from the dataset. Three identified fosmids were completely sequenced. The cloned genomic fragments within these fosmids are closely related to members of the Spirochaetaceae, Bacteroidales and Firmicutes, and their FeFe-hydrogenase sequences are characterized by the structure type M3, which is common to clostridial enzymes. FeFe-hydrogenase sequences found in this study represent hitherto undetected sequences, indicating the high genetic diversity regarding these enzymes in MWWTP. Results suggest that MWWTP have to be considered as reservoirs for new FeFe-hydrogenase genes. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Pyrosequencing-based quantitative measurement of CALR mutation allele burdens and their clinical implications in patients with myeloproliferative neoplasms.

PubMed

Oh, Yejin; Song, Ik-Chan; Kim, Jimyung; Kwon, Gye Cheol; Koo, Sun Hoe; Kim, Seon Young

2018-05-01

We developed a pyrosequencing-based method for the quantification of CALR mutations and compared the results using Sanger sequencing, fragment length analysis (FLA), digital-droplet PCR (ddPCR), and next-generation sequencing (NGS). Method validation studies were performed using cloned plasmid controls. Samples from 24 patients with myeloproliferative neoplasms were evaluated. Among the 24 patients, 15 had CALR mutations (7 type 1, 2 type 2, and 6 other mutations). The type 1 or type 2 mutation-positive results from pyrosequencing exhibited 100% concordance with the Sanger sequencing results. One novel CALR mutation was not detected by pyrosequencing. The CALR mutation allele burdens measured by pyrosequencing were slightly lower than those measured by FLA but slightly higher than the results obtained using ddPCR. Pyrosequencing exhibited high correlations with both methods. The mutation allele burdens estimated by NGS were significantly lower than those measured by pyrosequencing. An increased CALR mutation allele burden was associated with overt primary myelofibrosis. Patients with >70% mutation allele burdens in myeloid cells had a significantly longer time from diagnosis (P = 0.007), more bone marrow fibrosis (P = 0.010), and lower hemoglobin (P = 0.007). Pyrosequencing was a useful rapid sequencing method to determine the burden of CALR mutations. Copyright © 2018 Elsevier B.V. All rights reserved.

Changes in soil bacterial community triggered by drought-induced gap succession preceded changes in soil C stocks and quality

PubMed Central

Yuste, Jorge Curiel; Barba, Josep; Fernandez-Gonzalez, Antonio José; Fernandez-Lopez, Manuel; Mattana, Stefania; Martinez-Vilalta, Jordi; Nolis, Pau; Lloret, Francisco

2012-01-01

The aim of this study was to understand how drought-induced tree mortality and subsequent secondary succession would affect soil bacterial taxonomic composition as well as soil organic matter (SOM) quantity and quality in a mixed Mediterranean forest where the Scots pine (Pinus sylvestris) population, affected by climatic drought-induced die-off, is being replaced by Holm-oaks (HO; Quercus ilex). We apply a high throughput DNA pyrosequencing technique and 13C solid-state Nuclear Magnetic Resonance (CP-MAS 13C NMR) to soils within areas of influence (defined as an surface with 2-m radius around the trunk) of different trees: healthy and affected (defoliated) pines, pines that died a decade ago and healthy HOs. Soil respiration was also measured in the same spots during a spring campaign using a static close-chamber method (soda lime). A decade after death, and before aerial colonization by the more competitive HOs have even taken place, we could not find changes in soil C pools (quantity and/or quality) associated with tree mortality and secondary succession. Unlike C pools, bacterial diversity and community structure were strongly determined by tree mortality. Convergence between the most abundant taxa of soil bacterial communities under dead pines and colonizer trees (HOs) further suggests that physical gap colonization was occurring below-ground before above-ground colonization was taken place. Significantly higher soil respiration rates under dead trees, together with higher bacterial diversity and anomalously high representation of bacteria commonly associated with copiotrophic environments (r-strategic bacteria) further gives indications of how drought-induced tree mortality and secondary succession were influencing the structure of microbial communities and the metabolic activity of soils. PMID:23301169

Microbial communities of biomethanization digesters fed with raw and heat pre-treated microalgae biomasses.

PubMed

Sanz, Jose Luis; Rojas, Patricia; Morato, Ana; Mendez, Lara; Ballesteros, Mercedes; González-Fernández, Cristina

2017-02-01

Microalgae biomasses are considered promising feedstocks for biofuel and methane productions. Two Continuously Stirred Tank Reactors (CSTR), fed with fresh (CSTR-C) and heat pre-treated (CSTR-T) Chlorella biomass were run in parallel in order to determine methane productions. The methane yield was 1.5 times higher in CSTR-T with regard to CSTR-C. Aiming to understand the microorganism roles within of the reactors, the sludge used as an inoculum (I), plus raw (CSTR-C) and heat pre-treated (CSTR-T) samples were analyzed by high-throughput pyrosequencing. The bacterial communities were dominated by Proteobacteria, Bacteroidetes, Chloroflexi and Firmicutes. Spirochaetae and Actinobacteria were only detected in sample I. Proteobacteria, mainly Alfaproteobacteria, were by far the dominant phylum within of the CSTR-C bioreactor. Many of the sequences retrieved were related to bacteria present in activated sludge treatment plants and they were absent after thermal pre-treatment. Most of the sequences affiliated to the Bacteroidetes were related to uncultured groups. Anaerolineaceae was the sole family found of the Chloroflexi phylum. All of the genera identified of the Firmicutes phylum carried out macromolecule hydrolysis and by-product fermentation. The proteolytic bacteria were prevalent over the saccharolytic microbes. The percentage of the proteolytic genera increased from the inoculum to the CSTR-T sample in a parallel fashion with an available protein increase owing to the high protein content of Chlorella. To relate the taxa identified by high-throughput sequencing to their functional roles remains a future challenge. Copyright © 2016 Elsevier Ltd. All rights reserved.

Microreactor Cells for High-Throughput X-ray Absorption Spectroscopy

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

Beesley, Angela; Tsapatsaris, Nikolaos; Weiher, Norbert

2007-01-19

High-throughput experimentation has been applied to X-ray Absorption spectroscopy as a novel route for increasing research productivity in the catalysis community. Suitable instrumentation has been developed for the rapid determination of the local structure in the metal component of precursors for supported catalysts. An automated analytical workflow was implemented that is much faster than traditional individual spectrum analysis. It allows the generation of structural data in quasi-real time. We describe initial results obtained from the automated high throughput (HT) data reduction and analysis of a sample library implemented through the 96 well-plate industrial standard. The results show that a fullymore » automated HT-XAS technology based on existing industry standards is feasible and useful for the rapid elucidation of geometric and electronic structure of materials.« less

Decreased Diversity of the Oral Microbiota of Patients with Hepatitis B Virus-Induced Chronic Liver Disease: A Pilot Project

PubMed Central

Ling, Zongxin; Liu, Xia; Cheng, Yiwen; Jiang, Xiawei; Jiang, Haiyin; Wang, Yuezhu; Li, Lanjuan

2015-01-01

Increasing evidence suggests that altered gut microbiota is implicated in the pathogenesis of hepatitis B virus-induced chronic liver disease (HBV-CLD). However, the structure and composition of the oral microbiota of patients with HBV-CLD remains unclear. High-throughput pyrosequencing showed that decreased oral bacterial diversity was found in patients with HBV-CLD. The Firmicutes/Bacteroidetes ratio was increased significantly, which indicated that dysbiosis of the oral microbiota participated in the process of HBV-CLD development. However, the changing patterns of the oral microbiota in patients with HBV-induced liver cirrhosis (LC) were almost similar to patients with chronic hepatitis B (CHB). HBV infection resulted in an increase in potential H2S- and CH3SH-producing phylotypes such as Fusobacterium, Filifactor, Eubacterium, Parvimonas and Treponema, which might contribute to the increased oral malodor. These key oral-derived phylotypes might invade into the gut as opportunistic pathogens and contribute to altering the composition of the gut microbiota. This study provided important clues that dysbiosis of the oral microbiota might be involved in the development of HBV-CLD. Greater understanding of the relationships between the dysbiosis of oral microbiota and the development of HBV-CLD might facilitate the development of non-invasive differential diagnostic procedures and targeted treatments of HBV-CLD patients harbouring specific oral phylotypes. PMID:26606973

Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing.

PubMed

Massana, Ramon; Gobet, Angélique; Audic, Stéphane; Bass, David; Bittner, Lucie; Boutte, Christophe; Chambouvet, Aurélie; Christen, Richard; Claverie, Jean-Michel; Decelle, Johan; Dolan, John R; Dunthorn, Micah; Edvardsen, Bente; Forn, Irene; Forster, Dominik; Guillou, Laure; Jaillon, Olivier; Kooistra, Wiebe H C F; Logares, Ramiro; Mahé, Frédéric; Not, Fabrice; Ogata, Hiroyuki; Pawlowski, Jan; Pernice, Massimo C; Probert, Ian; Romac, Sarah; Richards, Thomas; Santini, Sébastien; Shalchian-Tabrizi, Kamran; Siano, Raffaele; Simon, Nathalie; Stoeck, Thorsten; Vaulot, Daniel; Zingone, Adriana; de Vargas, Colomban

2015-10-01

Although protists are critical components of marine ecosystems, they are still poorly characterized. Here we analysed the taxonomic diversity of planktonic and benthic protist communities collected in six distant European coastal sites. Environmental deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from three size fractions (pico-, nano- and micro/mesoplankton), as well as from dissolved DNA and surface sediments were used as templates for tag pyrosequencing of the V4 region of the 18S ribosomal DNA. Beta-diversity analyses split the protist community structure into three main clusters: picoplankton-nanoplankton-dissolved DNA, micro/mesoplankton and sediments. Within each cluster, protist communities from the same site and time clustered together, while communities from the same site but different seasons were unrelated. Both DNA and RNA-based surveys provided similar relative abundances for most class-level taxonomic groups. Yet, particular groups were overrepresented in one of the two templates, such as marine alveolates (MALV)-I and MALV-II that were much more abundant in DNA surveys. Overall, the groups displaying the highest relative contribution were Dinophyceae, Diatomea, Ciliophora and Acantharia. Also, well represented were Mamiellophyceae, Cryptomonadales, marine alveolates and marine stramenopiles in the picoplankton, and Monadofilosa and basal Fungi in sediments. Our extensive and systematic sequencing of geographically separated sites provides the most comprehensive molecular description of coastal marine protist diversity to date. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

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

PubMed Central

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

High Throughput Light Absorber Discovery, Part 2: Establishing Structure-Band Gap Energy Relationships.

PubMed

Suram, Santosh K; Newhouse, Paul F; Zhou, Lan; Van Campen, Douglas G; Mehta, Apurva; Gregoire, John M

2016-11-14

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

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

DOE PAGES

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

2016-05-26

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

A pyrosequencing-based assay for the rapid detection of the 22q11.2 deletion in DNA from buccal and dried blood spot samples.

PubMed

Koontz, Deborah; Baecher, Kirsten; Kobrynski, Lisa; Nikolova, Stanimila; Gallagher, Margaret

2014-09-01

The 22q11.2 deletion syndrome is one of the most common deletion syndromes in newborns. Some affected newborns may be diagnosed shortly after birth because of the presence of heart defects, palatal defects, or severe immune deficiencies. However, diagnosis is often delayed in patients presenting with other associated conditions that would benefit from early recognition and treatment, such as speech delays, learning difficulties, and schizophrenia. Fluorescence in situ hybridization (FISH) is the gold standard for deletion detection, but it is costly and time consuming and requires a whole blood specimen. Our goal was to develop a suitable assay for population-based screening of easily collectible specimens, such as buccal swabs and dried blood spots (DBS). We designed a pyrosequencing assay and validated it using DNA from FISH-confirmed 22q11 deletion syndrome patients and normal controls. We tested DBS from nine patients and paired buccal cell and venous blood specimens from 20 patients. Results were 100% concordant with FISH assay results. DNA samples from normal controls (n = 180 cell lines, n = 15 DBS, and n = 88 buccal specimens) were negative for the deletion. Limiting dilution experiments demonstrated that accurate results could be obtained from as little as 1 ng of DNA. This method represents a reliable and low-cost alternative for detection of the common 22q11.2 microdeletions and can be adapted to high-throughput population screening. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Abiotic factors shape microbial diversity in Sonoran Desert soils.

PubMed

Andrew, David R; Fitak, Robert R; Munguia-Vega, Adrian; Racolta, Adriana; Martinson, Vincent G; Dontsova, Katerina

2012-11-01

High-throughput, culture-independent surveys of bacterial and archaeal communities in soil have illuminated the importance of both edaphic and biotic influences on microbial diversity, yet few studies compare the relative importance of these factors. Here, we employ multiplexed pyrosequencing of the 16S rRNA gene to examine soil- and cactus-associated rhizosphere microbial communities of the Sonoran Desert and the artificial desert biome of the Biosphere2 research facility. The results of our replicate sampling approach show that microbial communities are shaped primarily by soil characteristics associated with geographic locations, while rhizosphere associations are secondary factors. We found little difference between rhizosphere communities of the ecologically similar saguaro (Carnegiea gigantea) and cardón (Pachycereus pringlei) cacti. Both rhizosphere and soil communities were dominated by the disproportionately abundant Crenarchaeota class Thermoprotei, which comprised 18.7% of 183,320 total pyrosequencing reads from a comparatively small number (1,337 or 3.7%) of the 36,162 total operational taxonomic units (OTUs). OTUs common to both soil and rhizosphere samples comprised the bulk of raw sequence reads, suggesting that the shared community of soil and rhizosphere microbes constitute common and abundant taxa, particularly in the bacterial phyla Proteobacteria, Actinobacteria, Planctomycetes, Firmicutes, Bacteroidetes, Chloroflexi, and Acidobacteria. The vast majority of OTUs, however, were rare and unique to either soil or rhizosphere communities and differed among locations dozens of kilometers apart. Several soil properties, particularly soil pH and carbon content, were significantly correlated with community diversity measurements. Our results highlight the importance of culture-independent approaches in surveying microbial communities of extreme environments.

Abiotic Factors Shape Microbial Diversity in Sonoran Desert Soils

PubMed Central

Fitak, Robert R.; Munguia-Vega, Adrian; Racolta, Adriana; Martinson, Vincent G.; Dontsova, Katerina

2012-01-01

High-throughput, culture-independent surveys of bacterial and archaeal communities in soil have illuminated the importance of both edaphic and biotic influences on microbial diversity, yet few studies compare the relative importance of these factors. Here, we employ multiplexed pyrosequencing of the 16S rRNA gene to examine soil- and cactus-associated rhizosphere microbial communities of the Sonoran Desert and the artificial desert biome of the Biosphere2 research facility. The results of our replicate sampling approach show that microbial communities are shaped primarily by soil characteristics associated with geographic locations, while rhizosphere associations are secondary factors. We found little difference between rhizosphere communities of the ecologically similar saguaro (Carnegiea gigantea) and cardón (Pachycereus pringlei) cacti. Both rhizosphere and soil communities were dominated by the disproportionately abundant Crenarchaeota class Thermoprotei, which comprised 18.7% of 183,320 total pyrosequencing reads from a comparatively small number (1,337 or 3.7%) of the 36,162 total operational taxonomic units (OTUs). OTUs common to both soil and rhizosphere samples comprised the bulk of raw sequence reads, suggesting that the shared community of soil and rhizosphere microbes constitute common and abundant taxa, particularly in the bacterial phyla Proteobacteria, Actinobacteria, Planctomycetes, Firmicutes, Bacteroidetes, Chloroflexi, and Acidobacteria. The vast majority of OTUs, however, were rare and unique to either soil or rhizosphere communities and differed among locations dozens of kilometers apart. Several soil properties, particularly soil pH and carbon content, were significantly correlated with community diversity measurements. Our results highlight the importance of culture-independent approaches in surveying microbial communities of extreme environments. PMID:22885757

High-throughput continuous hydrothermal synthesis of nanomaterials (part II): unveiling the as-prepared CexZryYzO2-δ phase diagram.

PubMed

Quesada-Cabrera, Raul; Weng, Xiaole; Hyett, Geoff; Clark, Robin J H; Wang, Xue Z; Darr, Jawwad A

2013-09-09

High-throughput continuous hydrothermal flow synthesis was used to manufacture 66 unique nanostructured oxide samples in the Ce-Zr-Y-O system. This synthesis approach resulted in a significant increase in throughput compared to that of conventional batch or continuous hydrothermal synthesis methods. The as-prepared library samples were placed into a wellplate for both automated high-throughput powder X-ray diffraction and Raman spectroscopy data collection, which allowed comprehensive structural characterization and phase mapping. The data suggested that a continuous cubic-like phase field connects all three Ce-Zr-O, Ce-Y-O, and Y-Zr-O binary systems together with a smooth and steady transition between the structures of neighboring compositions. The continuous hydrothermal process led to as-prepared crystallite sizes in the range of 2-7 nm (as determined by using the Scherrer equation).

Bacterial Communities in the Rhizospheres of Three Mangrove Tree Species from Beilun Estuary, China.

PubMed

Wu, Peng; Xiong, Xiaofei; Xu, Zhanzhou; Lu, Chuqian; Cheng, Hao; Lyu, Xiangli; Zhang, Jinghuai; He, Wei; Deng, Wei; Lyu, Yihua; Lou, Quansheng; Hong, Yiguo; Fang, Hongda

2016-01-01

The bacterial communities played important roles in the high productivity mangrove ecosystem. In this study, we investigated the vertical distributions of rhizosphere bacteria from three mangrove species (Bruguiera gymnorrhiza, Kandelia candel and Aegiceras corniculatum) in Beilun Estuary, China using high throughput DNA pyrosequencing of the 16S rRNA gene. Phylogenetic analysis showed that bacterial communities from mangrove rhizosphere sediments were dominated by Proteobacteria (mostly Deltaproteobacteria and Gammaproteobacteria), followed by Chloroflexi, Bacteroidetes, Planctomycetes and Acidobacteria. However, the ANOVA analysis on Shannon and Chao1 indices indicated that bacterial communities among sediments of the three mangrove species varied more strongly than the sampling depths. In addition, the PCA result demonstrated that the bacterial communities could be separated into three groups according to the mangrove species. Moreover, the dominated orders Rhodospirillales, GCA004 and envOPS12 were significantly different among sediments of the three mangrove species. The results of this study provided valuable information about the distribution feature of rhizosphere bacteria from Chinese mangrove plants and shed insights into biogeochemical transformations driven by bacteria in rhizosphere sediments.

Bacterial Communities in the Rhizospheres of Three Mangrove Tree Species from Beilun Estuary, China

PubMed Central

Wu, Peng; Xiong, Xiaofei; Xu, Zhanzhou; Lu, Chuqian; Cheng, Hao; Lyu, Xiangli; Zhang, Jinghuai; He, Wei; Deng, Wei; Lyu, Yihua; Lou, Quansheng; Hong, Yiguo; Fang, Hongda

2016-01-01

The bacterial communities played important roles in the high productivity mangrove ecosystem. In this study, we investigated the vertical distributions of rhizosphere bacteria from three mangrove species (Bruguiera gymnorrhiza, Kandelia candel and Aegiceras corniculatum) in Beilun Estuary, China using high throughput DNA pyrosequencing of the 16S rRNA gene. Phylogenetic analysis showed that bacterial communities from mangrove rhizosphere sediments were dominated by Proteobacteria (mostly Deltaproteobacteria and Gammaproteobacteria), followed by Chloroflexi, Bacteroidetes, Planctomycetes and Acidobacteria. However, the ANOVA analysis on Shannon and Chao1 indices indicated that bacterial communities among sediments of the three mangrove species varied more strongly than the sampling depths. In addition, the PCA result demonstrated that the bacterial communities could be separated into three groups according to the mangrove species. Moreover, the dominated orders Rhodospirillales, GCA004 and envOPS12 were significantly different among sediments of the three mangrove species. The results of this study provided valuable information about the distribution feature of rhizosphere bacteria from Chinese mangrove plants and shed insights into biogeochemical transformations driven by bacteria in rhizosphere sediments. PMID:27695084

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

PubMed

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

2009-01-01

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

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

NASA Technical Reports Server (NTRS)

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

2005-01-01

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

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

NASA Technical Reports Server (NTRS)

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

2004-01-01

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

The arbuscular mycorrhizal fungi colonising roots and root nodules of New Zealand kauri Agathis australis.

PubMed

Padamsee, Mahajabeen; Johansen, Renee B; Stuckey, S Alexander; Williams, Stephen E; Hooker, John E; Burns, Bruce R; Bellgard, Stanley E

2016-05-01

As the only endemic member in New Zealand of the ancient conifer family, Araucariaceae, Agathis australis is an ideal species to study putatively long-evolved mycorrhizal symbioses. However, little is known about A. australis root and nodular arbuscular mycorrhizal fungi (AMF), and how mycorrhizal colonisation occurs. We used light, scanning and transmission electron microscopy to characterise colonisation, and 454-sequencing to identify the AMF associated with A. australis roots and nodules. We interpreted the results in terms of the edaphic characteristics of the A. australis-influenced ecosystem. Representatives of five families of Glomeromycota were identified via high-throughput pyrosequencing. Imaging studies showed that there is abundant, but not ubiquitous, colonisation of nodules, which suggests that nodules are mostly colonised by horizontal transmission. Roots were also found to harbour AMF. This study is the first to demonstrate the multiple Glomeromycota lineages associated with A. australis including some that may not have been previously detected. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Comparing activated carbon of different particle sizes on enhancing methane generation in upflow anaerobic digester.

PubMed

Xu, Suyun; He, Chuanqiu; Luo, Liwen; Lü, Fan; He, Pinjing; Cui, Lifeng

2015-11-01

Two sizes of conductive particles, i.e. 10-20 mesh granulated activated carbon (GAC) and 80-100 mesh powdered activated carbon (PAC) were added into lab-scale upflow anaerobic sludge blanket reactors, respectively, to testify their enhancement on the syntrophic metabolism of alcohols and volatile fatty acids (VFAs) in 95days operation. When OLR increased to more than 5.8gCOD/L/d, the differences between GAC/PAC supplemented reactors and the control reactor became more significant. The introduction of activated carbon could facilitate the enrichment of methanogens and accelerate the startup of methanogenesis, as indicated by enhanced methane yield and substrate degradation. High-throughput pyrosequencing analysis showed that syntrophic bacteria and Methanosarcina sp. with versatile metabolic capability increased in the tightly absorbed fraction on the PAC surface, leading to the promoted syntrophic associations. Thus PAC prevails over than GAC for methanogenic reactor with heavy load. Copyright © 2015 Elsevier Ltd. All rights reserved.

Enhanced microbial reduction of vanadium (V) in groundwater with bioelectricity from microbial fuel cells

NASA Astrophysics Data System (ADS)

Hao, Liting; Zhang, Baogang; Tian, Caixing; Liu, Ye; Shi, Chunhong; Cheng, Ming; Feng, Chuanping

2015-08-01

Bioelectricity generated from the microbial fuel cell (MFC) is applied to the bioelectrical reactor (BER) directly to enhance microbial reduction of vanadium (V) (V(V)) in groundwater. With the maximum power density of 543.4 mW m-2 from the MFC, V(V) removal is accelerated with efficiency of 93.6% during 12 h operation. Higher applied voltage can facilitate this process. V(V) removals decrease with the increase of initial V(V) concentration, while extra addition of chemical oxygen demand (COD) has little effect on performance improvement. Microbial V(V) reduction is enhanced and then suppressed with the increase of conductivity. High-throughput 16S rRNA gene pyrosequencing analysis implies the accumulated Enterobacter and Lactococcus reduce V(V) with products from fermentative microorganisms such as Macellibacteroides. The presentation of electrochemically active bacteria as Enterobacter promotes electron transfers. This study indicates that application of bioelectricity from MFCs is a promising strategy to improve the efficiency of in-situ bioremediation of V(V) polluted groundwater.

Bacterial diversity is strongly associated with historical penguin activity in an Antarctic lake sediment profile.

PubMed

Zhu, Renbin; Shi, Yu; Ma, Dawei; Wang, Can; Xu, Hua; Chu, Haiyan

2015-11-25

Current penguin activity in Antarctica affects the geochemistry of sediments and their microbial communities; the effects of historical penguin activity are less well understood. Here, bacterial diversity in ornithogenic sediment was investigated using high-throughput pyrosequencing. The relative abundances of dominant phyla were controlled by the amount of historical penguin guano deposition. Significant positive correlations were found between both the bacterial richness and diversity, and the relative penguin number (p < 0.01); this indicated that historical penguin activity drove the vertical distribution of the bacterial communities. The lowest relative abundances of individual phyla corresponded to lowest number of penguin population at 1,800-2,300 yr BP during a drier and colder period; the opposite was observed during a moister and warmer climate (1,400-1,800 yr BP). This study shows that changes in the climate over millennia affected penguin populations and the outcomes of these changes affect the sediment bacterial community today.

Bacterial diversity is strongly associated with historical penguin activity in an Antarctic lake sediment profile

PubMed Central

Zhu, Renbin; Shi, Yu; Ma, Dawei; Wang, Can; Xu, Hua; Chu, Haiyan

2015-01-01

Current penguin activity in Antarctica affects the geochemistry of sediments and their microbial communities; the effects of historical penguin activity are less well understood. Here, bacterial diversity in ornithogenic sediment was investigated using high-throughput pyrosequencing. The relative abundances of dominant phyla were controlled by the amount of historical penguin guano deposition. Significant positive correlations were found between both the bacterial richness and diversity, and the relative penguin number (p < 0.01); this indicated that historical penguin activity drove the vertical distribution of the bacterial communities. The lowest relative abundances of individual phyla corresponded to lowest number of penguin population at 1,800–2,300 yr BP during a drier and colder period; the opposite was observed during a moister and warmer climate (1,400–1,800 yr BP). This study shows that changes in the climate over millennia affected penguin populations and the outcomes of these changes affect the sediment bacterial community today. PMID:26601753

Analysis of Nitrification Efficiency and Microbial Community in a Membrane Bioreactor Fed with Low COD/N-Ratio Wastewater

PubMed Central

Ma, Jinxing; Wang, Zhiwei; Zhu, Chaowei; Liu, Shumeng; Wang, Qiaoying; Wu, Zhichao

2013-01-01

In this study, an approach using influent COD/N ratio reduction was employed to improve process performance and nitrification efficiency in a membrane bioreactor (MBR). Besides sludge reduction, membrane fouling alleviation was observed during 330 d operation, which was attributed to the decreased production of soluble microbial products (SMP) and efficient carbon metabolism in the autotrophic nitrifying community. 454 high-throughput 16S rRNA gene pyrosequencing revealed that the diversity of microbial sequences was mainly determined by the feed characteristics, and that microbes could derive energy by switching to a more autotrophic metabolism to resist the environmental stress. The enrichment of nitrifiers in an MBR with a low COD/N-ratio demonstrated that this condition stimulated nitrification, and that the community distribution of ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) resulted in faster nitrite uptake rates. Further, ammonia oxidation was the rate-limiting step during the full nitrification. PMID:23667573

Microbial vanadium (V) reduction in groundwater with different soils from vanadium ore mining areas.

PubMed

Hao, Liting; Zhang, Baogang; Feng, Chuanping; Zhang, Zhenya; Lei, Zhongfang; Shimizu, Kazuya; Cao, Xuelong; Liu, Hui; Liu, Huipeng

2018-07-01

This work investigated the potential of vanadium (V) (V(V)) bioreduction by using soils sampled from four main kinds of vanadium ore mining areas, i.e. vanadium titanomagnetite, stone coal, petroleum associated minerals and uvanite as inocula. During a typical operation cycle of 60 h, the soils from vanadium titanomagnetite area and petroleum associated minerals area exhibited higher V(V) removal efficiencies, about 92.0 ± 2.0% and 91.0 ± 1.9% in comparison to 87.1 ± 1.9% and 69.0 ± 1.1% for the soils from uvanite and stone coal areas, respectively. Results from high-throughput 16 S rRNA gene pyrosequencing analysis reflect the accumulation of Bryobacter and Acidobacteriaceae with capabilities of V(V) reduction, accompanied with other functional species. This study is helpful to search new functional species for V(V) reduction and to develop in situ bioremediations of V(V) polluted groundwater. Copyright © 2018 Elsevier Ltd. All rights reserved.

Comparison of bacterial communities of conventional and A-stage activated sludge systems

PubMed Central

Gonzalez-Martinez, Alejandro; Rodriguez-Sanchez, Alejandro; Lotti, Tommaso; Garcia-Ruiz, Maria-Jesus; Osorio, Francisco; Gonzalez-Lopez, Jesus; van Loosdrecht, Mark C. M.

2016-01-01

The bacterial community structure of 10 different wastewater treatment systems and their influents has been investigated through pyrosequencing, yielding a total of 283486 reads. These bioreactors had different technological configurations: conventional activated sludge (CAS) systems and very highly loaded A-stage systems. A-stage processes are proposed as the first step in an energy producing municipal wastewater treatment process. Pyrosequencing analysis indicated that bacterial community structure of all influents was similar. Also the bacterial community of all CAS bioreactors was similar. Bacterial community structure of A-stage bioreactors showed a more case-specific pattern. A core of genera was consistently found for all influents, all CAS bioreactors and all A-stage bioreactors, respectively, showing that different geographical locations in The Netherlands and Spain did not affect the functional bacterial communities in these technologies. The ecological roles of these bacteria were discussed. Influents and A-stage bioreactors shared several core genera, while none of these were shared with CAS bioreactors communities. This difference is thought to reside in the different operational conditions of the two technologies. This study shows that bacterial community structure of CAS and A-stage bioreactors are mostly driven by solids retention time (SRT) and hydraulic retention time (HRT), as suggested by multivariate redundancy analysis. PMID:26728449

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

PubMed

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

2018-05-25

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

Characterization of the fecal microbiome in different swine groups by high-throughput sequencing.

PubMed

Park, Soo-Je; Kim, Jinu; Lee, Jong-Soo; Rhee, Sung-Keun; Kim, Hongik

2014-08-01

Swine have a complex microbial community within their gastrointestinal tract that plays a critical role in both health and disease. High-throughput 16S rRNA gene-based pyrosequencing was used to identify the possible core microorganisms in the gut of swine groups that differ in meat quality and weight grades (level 1 as higher meat quality and level 2 as lower meat quality). Samples were taken from the rectum and/or stool from ten animals, DNA was extracted, and the V1-V3 regions of the 16S rRNA gene were amplified. Two bacterial populations (Bacteroidetes and Firmicutes) dominated and were shared between the two groups. Significant differences between the groups were found at the genus level. The genera Lactobacillus and Oscillibacter were found in slightly higher proportions in the level 2 group (12.6 and 12.4% of the classified reads, respectively) than those of level 1 (9.6 and 7.7%, respectively). By contrast, the proportion of reads assigned to the genus Roseburia in the level 1 group (13.0%) was higher than that of level 2 (4.8%). The largest differences were related to the genera Clostridium, Oscillibacter, and Roseburia as core microorganisms. Moreover, two genera, Roseburia and Clostridium, related to level 1 produced linoleic acid or short chain fatty acids that might contribute to swine health and development. In conclusion, the presence of core bacteria in the swine gut is associated with meat quality with reduced body fat in swine. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

PubMed

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

2017-08-07

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

High-throughput sequence analysis of Ciona intestinalis SL trans-spliced mRNAs: alternative expression modes and gene function correlates.

PubMed

Matsumoto, Jun; Dewar, Ken; Wasserscheid, Jessica; Wiley, Graham B; Macmil, Simone L; Roe, Bruce A; Zeller, Robert W; Satou, Yutaka; Hastings, Kenneth E M

2010-05-01

Pre-mRNA 5' spliced-leader (SL) trans-splicing occurs in some metazoan groups but not in others. Genome-wide characterization of the trans-spliced mRNA subpopulation has not yet been reported for any metazoan. We carried out a high-throughput analysis of the SL trans-spliced mRNA population of the ascidian tunicate Ciona intestinalis by 454 Life Sciences (Roche) pyrosequencing of SL-PCR-amplified random-primed reverse transcripts of tailbud embryo RNA. We obtained approximately 250,000 high-quality reads corresponding to 8790 genes, approximately 58% of the Ciona total gene number. The great depth of this data revealed new aspects of trans-splicing, including the existence of a significant class of "infrequently trans-spliced" genes, accounting for approximately 28% of represented genes, that generate largely non-trans-spliced mRNAs, but also produce trans-spliced mRNAs, in part through alternative promoter use. Thus, the conventional qualitative dichotomy of trans-spliced versus non-trans-spliced genes should be supplanted by a more accurate quantitative view recognizing frequently and infrequently trans-spliced gene categories. Our data include reads representing approximately 80% of Ciona frequently trans-spliced genes. Our analysis also revealed significant use of closely spaced alternative trans-splice acceptor sites which further underscores the mechanistic similarity of cis- and trans-splicing and indicates that the prevalence of +/-3-nt alternative splicing events at tandem acceptor sites, NAGNAG, is driven by spliceosomal mechanisms, and not nonsense-mediated decay, or selection at the protein level. The breadth of gene representation data enabled us to find new correlations between trans-splicing status and gene function, namely the overrepresentation in the frequently trans-spliced gene class of genes associated with plasma/endomembrane system, Ca(2+) homeostasis, and actin cytoskeleton.

High-throughput sequencing of retrotransposon integration provides a saturated profile of target activity in Schizosaccharomyces pombe.

PubMed

Guo, Yabin; Levin, Henry L

2010-02-01

The biological impact of transposons on the physiology of the host depends greatly on the frequency and position of integration. Previous studies of Tf1, a long terminal repeat retrotransposon in Schizosaccharomyces pombe, showed that integration occurs at the promoters of RNA polymerase II (Pol II) transcribed genes. To determine whether specific promoters are preferred targets of integration, we sequenced large numbers of insertions using high-throughput pyrosequencing. In four independent experiments we identified a total of 73,125 independent integration events. These data provided strong support for the conclusion that Pol II promoters are the targets of Tf1 integration. The size and number of the integration experiments resulted in reproducible measures of integration for each intergenic region and ORF in the S. pombe genome. The reproducibility of the integration activity from experiment to experiment demonstrates that we have saturated the full set of insertion sites that are actively targeted by Tf1. We found Tf1 integration was highly biased in favor of a specific set of Pol II promoters. The overwhelming majority (76%) of the insertions were distributed in intergenic sequences that contained 31% of the promoters of S. pombe. Interestingly, there was no correlation between the amount of integration at these promoters and their level of transcription. Instead, we found Tf1 had a strong preference for promoters that are induced by conditions of stress. This targeting of stress response genes coupled with the ability of Tf1 to regulate the expression of adjacent genes suggests Tf1 may improve the survival of S. pombe when cells are exposed to environmental stress.

High-throughput sequencing of retrotransposon integration provides a saturated profile of target activity in Schizosaccharomyces pombe

PubMed Central

Guo, Yabin; Levin, Henry L.

2010-01-01

The biological impact of transposons on the physiology of the host depends greatly on the frequency and position of integration. Previous studies of Tf1, a long terminal repeat retrotransposon in Schizosaccharomyces pombe, showed that integration occurs at the promoters of RNA polymerase II (Pol II) transcribed genes. To determine whether specific promoters are preferred targets of integration, we sequenced large numbers of insertions using high-throughput pyrosequencing. In four independent experiments we identified a total of 73,125 independent integration events. These data provided strong support for the conclusion that Pol II promoters are the targets of Tf1 integration. The size and number of the integration experiments resulted in reproducible measures of integration for each intergenic region and ORF in the S. pombe genome. The reproducibility of the integration activity from experiment to experiment demonstrates that we have saturated the full set of insertion sites that are actively targeted by Tf1. We found Tf1 integration was highly biased in favor of a specific set of Pol II promoters. The overwhelming majority (76%) of the insertions were distributed in intergenic sequences that contained 31% of the promoters of S. pombe. Interestingly, there was no correlation between the amount of integration at these promoters and their level of transcription. Instead, we found Tf1 had a strong preference for promoters that are induced by conditions of stress. This targeting of stress response genes coupled with the ability of Tf1 to regulate the expression of adjacent genes suggests Tf1 may improve the survival of S. pombe when cells are exposed to environmental stress. PMID:20040583

Seasonal diversity and dynamics of haptophytes in the Skagerrak, Norway, explored by high-throughput sequencing

PubMed Central

Egge, Elianne Sirnæs; Johannessen, Torill Vik; Andersen, Tom; Eikrem, Wenche; Bittner, Lucie; Larsen, Aud; Sandaa, Ruth-Anne; Edvardsen, Bente

2015-01-01

Microalgae in the division Haptophyta play key roles in the marine ecosystem and in global biogeochemical processes. Despite their ecological importance, knowledge on seasonal dynamics, community composition and abundance at the species level is limited due to their small cell size and few morphological features visible under the light microscope. Here, we present unique data on haptophyte seasonal diversity and dynamics from two annual cycles, with the taxonomic resolution and sampling depth obtained with high-throughput sequencing. From outer Oslofjorden, S Norway, nano- and picoplanktonic samples were collected monthly for 2 years, and the haptophytes targeted by amplification of RNA/cDNA with Haptophyta-specific 18S rDNA V4 primers. We obtained 156 operational taxonomic units (OTUs), from c. 400.000 454 pyrosequencing reads, after rigorous bioinformatic filtering and clustering at 99.5%. Most OTUs represented uncultured and/or not yet 18S rDNA-sequenced species. Haptophyte OTU richness and community composition exhibited high temporal variation and significant yearly periodicity. Richness was highest in September–October (autumn) and lowest in April–May (spring). Some taxa were detected all year, such as Chrysochromulina simplex, Emiliania huxleyi and Phaeocystis cordata, whereas most calcifying coccolithophores only appeared from summer to early winter. We also revealed the seasonal dynamics of OTUs representing putative novel classes (clades HAP-3–5) or orders (clades D, E, F). Season, light and temperature accounted for 29% of the variation in OTU composition. Residual variation may be related to biotic factors, such as competition and viral infection. This study provides new, in-depth knowledge on seasonal diversity and dynamics of haptophytes in North Atlantic coastal waters. PMID:25893259

Seasonal diversity and dynamics of haptophytes in the Skagerrak, Norway, explored by high-throughput sequencing.

PubMed

Egge, Elianne Sirnaes; Johannessen, Torill Vik; Andersen, Tom; Eikrem, Wenche; Bittner, Lucie; Larsen, Aud; Sandaa, Ruth-Anne; Edvardsen, Bente

2015-06-01

Microalgae in the division Haptophyta play key roles in the marine ecosystem and in global biogeochemical processes. Despite their ecological importance, knowledge on seasonal dynamics, community composition and abundance at the species level is limited due to their small cell size and few morphological features visible under the light microscope. Here, we present unique data on haptophyte seasonal diversity and dynamics from two annual cycles, with the taxonomic resolution and sampling depth obtained with high-throughput sequencing. From outer Oslofjorden, S Norway, nano- and picoplanktonic samples were collected monthly for 2 years, and the haptophytes targeted by amplification of RNA/cDNA with Haptophyta-specific 18S rDNA V4 primers. We obtained 156 operational taxonomic units (OTUs), from c. 400.000 454 pyrosequencing reads, after rigorous bioinformatic filtering and clustering at 99.5%. Most OTUs represented uncultured and/or not yet 18S rDNA-sequenced species. Haptophyte OTU richness and community composition exhibited high temporal variation and significant yearly periodicity. Richness was highest in September-October (autumn) and lowest in April-May (spring). Some taxa were detected all year, such as Chrysochromulina simplex, Emiliania huxleyi and Phaeocystis cordata, whereas most calcifying coccolithophores only appeared from summer to early winter. We also revealed the seasonal dynamics of OTUs representing putative novel classes (clades HAP-3-5) or orders (clades D, E, F). Season, light and temperature accounted for 29% of the variation in OTU composition. Residual variation may be related to biotic factors, such as competition and viral infection. This study provides new, in-depth knowledge on seasonal diversity and dynamics of haptophytes in North Atlantic coastal waters. © 2015 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.

Probing the Rare Biosphere of the North-West Mediterranean Sea: An Experiment with High Sequencing Effort.

PubMed

Crespo, Bibiana G; Wallhead, Philip J; Logares, Ramiro; Pedrós-Alió, Carlos

2016-01-01

High-throughput sequencing (HTS) techniques have suggested the existence of a wealth of species with very low relative abundance: the rare biosphere. We attempted to exhaustively map this rare biosphere in two water samples by performing an exceptionally deep pyrosequencing analysis (~500,000 final reads per sample). Species data were derived by a 97% identity criterion and various parametric distributions were fitted to the observed counts. Using the best-fitting Sichel distribution we estimate a total species richness of 1,568-1,669 (95% Credible Interval) and 5,027-5,196 for surface and deep water samples respectively, implying that 84-89% of the total richness in those two samples was sequenced, and we predict that a quadrupling of the present sequencing effort would suffice to observe 90% of the total richness in both samples. Comparing the HTS results with a culturing approach we found that most of the cultured taxa were not obtained by HTS, despite the high sequencing effort. Culturing therefore remains a useful tool for uncovering marine bacterial diversity, in addition to its other uses for studying the ecology of marine bacteria.

[Effect of X-ray micro-computed tomography on the metabolic activity and diversity of soil microbial communities in two Chinese soils].

PubMed

Zu, Qianhui; Fang, Huan; Zhou, Hu; Zhang, Jianwei; Peng, Xinhua; Lin, Xiangui; Feng, Youzhi

2016-01-04

X-ray micro-computed tomography (micro-CT) technology, as used in the in situ and nondestructive analysis of soil physical structure, provides the opportunity of associating soil physical and biological assays. Due to the high heterogeneity of the soil matrix, X-ray micro-CT scanning and soil microbial assays should be conducted on the same soil sample. This raises the question whether X-ray micro-CT influences microbial function and diversity of the sample soil to be analyzed. To address this question, we used plate counting, microcalorimetry and pyrosequencing approaches to evaluate the effect of X-ray--at doses typically used in micro-CT--on soil microorganisms in a typical soil of North China Plain, Fluvo-aquic soil and in a typical soil of subtropical China, Ultisol soil, respectively. In both soils radiation decreased the number of viable soil bacteria and disturbed their thermogenic profiles. At DNA level, pyrosequencing revealed that alpha diversities of two soils biota were influenced in opposite ways, while beta diversity was not affected although the relative abundances of some guilds were changed. These findings indicate that the metabolically active aspects of soil biota are not compatible with X-ray micro-CT; while the beta molecular diversity based on pyrosequencing could be compatible.

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

PubMed

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.

Seasonal changes in the digesta-adherent rumen bacterial communities of dairy cattle grazing pasture.

PubMed

Noel, Samantha J; Attwood, Graeme T; Rakonjac, Jasna; Moon, Christina D; Waghorn, Garry C; Janssen, Peter H

2017-01-01

The complex microbiota that resides within the rumen is responsible for the break-down of plant fibre. The bacteria that attach to ingested plant matter within the rumen are thought to be responsible for initial fibre degradation. Most studies examining the ecology of this important microbiome only offer a 'snapshot' in time. We monitored the diversity of rumen bacteria in four New Zealand dairy cows, grazing a rye-grass and clover pasture over five consecutive seasons, using high throughput pyrosequencing of bacterial 16S rRNA genes. We chose to focus on the digesta-adherent bacterial community to learn more about the stability of this community over time. 16S rRNA gene sequencing showed a high level of bacterial diversity, totalling 1539 operational taxonomic units (OTUs, grouped at 96% sequence similarity) across all samples, and ranging from 653 to 926 OTUs per individual sample. The nutritive composition of the pasture changed with the seasons as did the production phase of the animals. Sequence analysis showed that, overall, the bacterial communities were broadly similar between the individual animals. The adherent bacterial community was strongly dominated by members of Firmicutes (82.1%), followed by Bacteroidetes (11.8%). This community differed between the seasons, returning to close to that observed in the same season one year later. These seasonal differences were only small, but were statistically significant (p < 0.001), and were probably due to the seasonal differences in the diet. These results demonstrate a general invariability of the ruminal bacterial community structure in these grazing dairy cattle.

Detection of large numbers of novel sequences in the metatranscriptomes of complex marine microbial communities.

PubMed

Gilbert, Jack A; Field, Dawn; Huang, Ying; Edwards, Rob; Li, Weizhong; Gilna, Paul; Joint, Ian

2008-08-22

Sequencing the expressed genetic information of an ecosystem (metatranscriptome) can provide information about the response of organisms to varying environmental conditions. Until recently, metatranscriptomics has been limited to microarray technology and random cloning methodologies. The application of high-throughput sequencing technology is now enabling access to both known and previously unknown transcripts in natural communities. We present a study of a complex marine metatranscriptome obtained from random whole-community mRNA using the GS-FLX Pyrosequencing technology. Eight samples, four DNA and four mRNA, were processed from two time points in a controlled coastal ocean mesocosm study (Bergen, Norway) involving an induced phytoplankton bloom producing a total of 323,161,989 base pairs. Our study confirms the finding of the first published metatranscriptomic studies of marine and soil environments that metatranscriptomics targets highly expressed sequences which are frequently novel. Our alternative methodology increases the range of experimental options available for conducting such studies and is characterized by an exceptional enrichment of mRNA (99.92%) versus ribosomal RNA. Analysis of corresponding metagenomes confirms much higher levels of assembly in the metatranscriptomic samples and a far higher yield of large gene families with >100 members, approximately 91% of which were novel. This study provides further evidence that metatranscriptomic studies of natural microbial communities are not only feasible, but when paired with metagenomic data sets, offer an unprecedented opportunity to explore both structure and function of microbial communities--if we can overcome the challenges of elucidating the functions of so many never-seen-before gene families.

Core gut microbiota in Jinhua pigs and its correlation with strain, farm and weaning age.

PubMed

Yang, Hua; Xiao, Yingping; Wang, Junjun; Xiang, Yun; Gong, Yujie; Wen, Xueting; Li, Defa

2018-05-01

Gut microbial diversity and the core microbiota of the Jinhua pig, which is a traditional, slow-growing Chinese breed with a high body-fat content, were examined from a total of 105 fecal samples collected from 6 groups of pigs at 3 weaning ages that originated from 2 strains and were raised on 3 different pig farms. The bacterial community was analyzed following high-throughput pyrosequencing of 16S rRNA genes, and the fecal concentrations of short-chain fatty acids (SCFAs) were measured by gas chromatograph. Our results showed that Firmicutes and Bacteroidetes were the dominant phyla, and Lactobacillus, Streptococcus, Clostridium, SMB53, and Bifidobacterium were the most abundant genera. Fifteen predominant genera present in every Jinhua pig sample constituted a phylogenetic core microbiota and included the probiotics Lactobacillus and Bifidobacterium, and the SCFA-producing bacteria Clostridium, Prevotella, Bacteroides, Coprococcus, Roseburia, Ruminococcus, Blautia, and Butyricicoccus. Comparisons of the microbiota compositions and SCFA concentrations across the 6 groups of pigs demonstrated that genetic background and weaning age affected the structure of the gut microbiota more significantly than the farm. The relative abundance of the core genera in the pigs, including Lactobacillus, Clostridium, Prevotella, Bacteroides, Roseburia, Ruminococcus, Blautia, and Butyricicoccus varied dramatically in pigs among the 2 origins and 3 weaning ages, while Oscillospira, Megasphaera, Parabacteroides, and Corynebacterium differed among pigs from different farms. Interestingly, there was a more significant influence of strain and weaning age than of rearing farm on the SCFA concentrations. Therefore, strain and weaning age appear to be the more important factors shaping the intestinal microbiome of pigs.

Seasonal changes in the digesta-adherent rumen bacterial communities of dairy cattle grazing pasture

PubMed Central

Attwood, Graeme T.; Rakonjac, Jasna; Moon, Christina D.; Waghorn, Garry C.; Janssen, Peter H.

2017-01-01

The complex microbiota that resides within the rumen is responsible for the break-down of plant fibre. The bacteria that attach to ingested plant matter within the rumen are thought to be responsible for initial fibre degradation. Most studies examining the ecology of this important microbiome only offer a ‘snapshot’ in time. We monitored the diversity of rumen bacteria in four New Zealand dairy cows, grazing a rye-grass and clover pasture over five consecutive seasons, using high throughput pyrosequencing of bacterial 16S rRNA genes. We chose to focus on the digesta-adherent bacterial community to learn more about the stability of this community over time. 16S rRNA gene sequencing showed a high level of bacterial diversity, totalling 1539 operational taxonomic units (OTUs, grouped at 96% sequence similarity) across all samples, and ranging from 653 to 926 OTUs per individual sample. The nutritive composition of the pasture changed with the seasons as did the production phase of the animals. Sequence analysis showed that, overall, the bacterial communities were broadly similar between the individual animals. The adherent bacterial community was strongly dominated by members of Firmicutes (82.1%), followed by Bacteroidetes (11.8%). This community differed between the seasons, returning to close to that observed in the same season one year later. These seasonal differences were only small, but were statistically significant (p < 0.001), and were probably due to the seasonal differences in the diet. These results demonstrate a general invariability of the ruminal bacterial community structure in these grazing dairy cattle. PMID:28296930

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

PubMed

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.

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

PubMed

Baldi, Pierre

2011-12-27

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

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

PubMed Central

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

Lactic acid fermentation from food waste with indigenous microbiota: Effects of pH, temperature and high OLR.

PubMed

Tang, Jialing; Wang, Xiaochang; Hu, Yisong; Zhang, Yongmei; Li, Yuyou

2016-06-01

The effects of pH, temperature and high organic loading rate (OLR) on lactic acid production from food waste without extra inoculum addition were investigated in this study. Using batch experiments, the results showed that although the hydrolysis rate increased with pH adjustment, the lactic acid concentration and productivity were highest at pH 6. High temperatures were suitable for solubilization but seriously restricted the acidification processes. The highest lactic acid yield (0.46g/g-TS) and productivity (278.1mg/Lh) were obtained at 37°C and pH 6. In addition, the lactic acid concentration gradually increased with the increase in OLR, and the semi-continuous reactor could be stably operated at an OLR of 18g-TS/Ld. However, system instability, low lactic acid yield and a decrease in VS removal were noticed at high OLRs (22g-TS/Ld). The concentrations of volatile fatty acids (VFAs) in the fermentation mixture were relatively low but slightly increased with OLR, and acetate was the predominant VFA component. Using high-throughput pyrosequencing, Lactobacillus from the raw food waste was found to selectively accumulate and become dominant in the semi-continuous reactor. Copyright © 2016 Elsevier Ltd. All rights reserved.

Collaborative Core Research Program for Chemical-Biological Warfare Defense

DTIC Science & Technology

2015-01-04

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

Searching for resistance genes to Bursaphelenchus xylophilus using high throughput screening.

PubMed

Santos, Carla S; Pinheiro, Miguel; Silva, Ana I; Egas, Conceição; Vasconcelos, Marta W

2012-11-07

Pine wilt disease (PWD), caused by the pinewood nematode (PWN; Bursaphelenchus xylophilus), damages and kills pine trees and is causing serious economic damage worldwide. Although the ecological mechanism of infestation is well described, the plant's molecular response to the pathogen is not well known. This is due mainly to the lack of genomic information and the complexity of the disease. High throughput sequencing is now an efficient approach for detecting the expression of genes in non-model organisms, thus providing valuable information in spite of the lack of the genome sequence. In an attempt to unravel genes potentially involved in the pine defense against the pathogen, we hereby report the high throughput comparative sequence analysis of infested and non-infested stems of Pinus pinaster (very susceptible to PWN) and Pinus pinea (less susceptible to PWN). Four cDNA libraries from infested and non-infested stems of P. pinaster and P. pinea were sequenced in a full 454 GS FLX run, producing a total of 2,083,698 reads. The putative amino acid sequences encoded by the assembled transcripts were annotated according to Gene Ontology, to assign Pinus contigs into Biological Processes, Cellular Components and Molecular Functions categories. Most of the annotated transcripts corresponded to Picea genes-25.4-39.7%, whereas a smaller percentage, matched Pinus genes, 1.8-12.8%, probably a consequence of more public genomic information available for Picea than for Pinus. The comparative transcriptome analysis showed that when P. pinaster was infested with PWN, the genes malate dehydrogenase, ABA, water deficit stress related genes and PAR1 were highly expressed, while in PWN-infested P. pinea, the highly expressed genes were ricin B-related lectin, and genes belonging to the SNARE and high mobility group families. Quantitative PCR experiments confirmed the differential gene expression between the two pine species. Defense-related genes triggered by nematode infestation were detected in both P. pinaster and P. pinea transcriptomes utilizing 454 pyrosequencing technology. P. pinaster showed higher abundance of genes related to transcriptional regulation, terpenoid secondary metabolism (including some with nematicidal activity) and pathogen attack. P. pinea showed higher abundance of genes related to oxidative stress and higher levels of expression in general of stress responsive genes. This study provides essential information about the molecular defense mechanisms utilized by P. pinaster and P. pinea against PWN infestation and contributes to a better understanding of PWD.

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

PubMed

Montecchia, Marcela S; Tosi, Micaela; Soria, Marcelo A; Vogrig, Jimena A; Sydorenko, Oksana; Correa, Olga S

2015-01-01

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.

Pyrosequencing Reveals Changes in Soil Bacterial Communities after Conversion of Yungas Forests to Agriculture

PubMed Central

Montecchia, Marcela S.; Tosi, Micaela; Soria, Marcelo A.; Vogrig, Jimena A.; Sydorenko, Oksana; Correa, Olga S.

2015-01-01

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. PMID:25793893

Urease gene-containing Archaea dominate autotrophic ammonia oxidation in two acid soils.

PubMed

Lu, Lu; Jia, Zhongjun

2013-06-01

The metabolic traits of ammonia-oxidizing archaea (AOA) and bacteria (AOB) interacting with their environment determine the nitrogen cycle at the global scale. Ureolytic metabolism has long been proposed as a mechanism for AOB to cope with substrate paucity in acid soil, but it remains unclear whether urea hydrolysis could afford AOA greater ecological advantages. By combining DNA-based stable isotope probing (SIP) and high-throughput pyrosequencing, here we show that autotrophic ammonia oxidation in two acid soils was predominately driven by AOA that contain ureC genes encoding the alpha subunit of a putative archaeal urease. In urea-amended SIP microcosms of forest soil (pH 5.40) and tea orchard soil (pH 3.75), nitrification activity was stimulated significantly by urea fertilization when compared with water-amended soils in which nitrification resulted solely from the oxidation of ammonia generated through mineralization of soil organic nitrogen. The stimulated activity was paralleled by changes in abundance and composition of archaeal amoA genes. Time-course incubations indicated that archaeal amoA genes were increasingly labelled by (13) CO2 in both microcosms amended with water and urea. Pyrosequencing revealed that archaeal populations were labelled to a much greater extent in soils amended with urea than water. Furthermore, archaeal ureC genes were successfully amplified in the (13) C-DNA, and acetylene inhibition suggests that autotrophic growth of urease-containing AOA depended on energy generation through ammonia oxidation. The sequences of AOB were not detected, and active AOA were affiliated with the marine Group 1.1a-associated lineage. The results suggest that ureolytic N metabolism could afford AOA greater advantages for autotrophic ammonia oxidation in acid soil, but the mechanism of how urea activates AOA cells remains unclear. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

High-throughput screening of dye-ligands for chromatography.

PubMed

Kumar, Sunil; Punekar, Narayan S

2014-01-01

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

Phylogenetic Characterization of Fecal Microbial Communities of Dogs Fed Diets with or without Supplemental Dietary Fiber Using 454 Pyrosequencing

PubMed Central

Middelbos, Ingmar S.; Vester Boler, Brittany M.; Qu, Ani; White, Bryan A.; Swanson, Kelly S.; Fahey, George C.

2010-01-01

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. PMID:20339542

High throughput platforms for structural genomics of integral membrane proteins.

PubMed

Mancia, Filippo; Love, James

2011-08-01

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

[Effect of long-term fertilizing regime on soil microbial diversity and soil property].

PubMed

Li, Chenhua; Zhang, Caixia; Tang, Lisong; Xiong, Zhengqin; Wang, Baozhan; Jia, Zhongjun; Li, Yan

2014-03-04

To evaluate the effect of long-term fertilization on soil microbial community and soil chemical and physical properties. Using a high-throughput pyrosequencing technique, we studied microbial community in the 0-300 cm soil samples covering a 20-year field-experiment with different fertilization applications including inorganic fertilizer alone (N 300 kg/hm2, P2O5 150 kg/hm2 and K2O 60 kg/hm2) and inorganic fertilizer combined with straw (same application rate of N and P fertilizer combined with 5.4 t straw). Actinobacteria and alpha-proteobacteria were the predominant groups in the topsoil (0-20 cm). As the soil depth increased, the relative abundance of actinobacteria decreased whereas that of proteobacteria, especially gamma-proteobacteria and beta-proteobacteria increased and gradually became the dominant groups in the subsoil (20-300 cm). Long-term fertilizing applications significantly affected soil microbial communities throughout the soil profile, and increased the relative abundance of ammonia-oxidizing archaea at 0-40 cm depth. In addition, agriculture management, e. g. irrigation may be an important driving factor for the distribution of ammonia-oxidizing bacteria in soil profile. Total nitrogen and organic carbon contents were the most influential factors on microbial community in the topsoil and in the subsoil, respectively. Long-term fertilizer applications altered soil nutrient availability within the soil profile, which was likely to result in the different microbial community structure between the fertilizer treatments, especially for the subsoil.

A long-term field experiment of soil transplantation demonstrating the role of contemporary geographic separation in shaping soil microbial community structure

PubMed Central

Sun, Bo; Wang, Feng; Jiang, Yuji; Li, Yun; Dong, Zhixin; Li, Zhongpei; Zhang, Xue-Xian

2014-01-01

The spatial patterns of microbial communities are largely determined by the combined effects of historical contingencies and contemporary environmental disturbances, but their relative importance remains poorly understood. Empirical biogeographic data currently available are mostly based on the traditional method of observational survey, which typically involves comparing indigenous microbial communities across spatial scales. Here, we report a long-term soil transplantation experiment, whereby the same two soils (red Acrisol and purple Cambisol from Yingtan) were placed into two geographic locations of ∼1000 km apart (i.e., Yingtan in the mid-subtropical region and Fengqiu in warm-temperate region; both located in China). Twenty years after the transplantation, the resulting soil microbial communities were subject to high-throughput 454 pyrosequencing analysis of 16S and 18S rRNA genes. Additionally, bacteria and archaea involved in nitrogen cycling were estimated using clone library analysis of four genes: archaeal amoA, bacterial amoA,nirK, and nifH. Data of subsequent phylogenetic analysis show that bacteria, fungi, and other microbial eukaryotes, as well as the nitrogen cycling genes, are grouped primarily by the factor of geographic location rather than soil type. Moreover, a shift of microbial communities toward those in local soil (i.e., Chao soil in Fengqiu) has been observed. The results thus suggest that the historical effects persistent in the soil microbial communities can be largely erased by contemporary disturbance within a short period of 20 years, implicating weak effects of historical contingencies on the structure and composition of microbial communities in the soil. PMID:24772284

Characterization of Metabolically Active Bacterial Populations in Subseafloor Nankai Trough Sediments above, within, and below the Sulfate–Methane Transition Zone

PubMed Central

Mills, Heath J.; Reese, Brandi Kiel; Shepard, Alicia K.; Riedinger, Natascha; Dowd, Scot E.; Morono, Yuki; Inagaki, Fumio

2012-01-01

A remarkable number of microbial cells have been enumerated within subseafloor sediments, suggesting a biological impact on geochemical processes in the subseafloor habitat. However, the metabolically active fraction of these populations is largely uncharacterized. In this study, an RNA-based molecular approach was used to determine the diversity and community structure of metabolically active bacterial populations in the upper sedimentary formation of the Nankai Trough seismogenic zone. Samples used in this study were collected from the slope apron sediment overlying the accretionary prism at Site C0004 during the Integrated Ocean Drilling Program Expedition 316. The sediments represented microbial habitats above, within, and below the sulfate–methane transition zone (SMTZ), which was observed approximately 20 m below the seafloor (mbsf). Small subunit ribosomal RNA were extracted, quantified, amplified, and sequenced using high-throughput 454 pyrosequencing, indicating the occurrence of metabolically active bacterial populations to a depth of 57 mbsf. Transcript abundance and bacterial diversity decreased with increasing depth. The two communities below the SMTZ were similar at the phylum level, however only a 24% overlap was observed at the genus level. Active bacterial community composition was not confined to geochemically predicted redox stratification despite the deepest sample being more than 50 m below the oxic/anoxic interface. Genus-level classification suggested that the metabolically active subseafloor bacterial populations had similarities to previously cultured organisms. This allowed predictions of physiological potential, expanding understanding of the subseafloor microbial ecosystem. Unique community structures suggest very diverse active populations compared to previous DNA-based diversity estimates, providing more support for enhancing community characterizations using more advanced sequencing techniques. PMID:22485111

Intracellular diversity of the V4 and V9 regions of the 18S rRNA in marine protists (radiolarians) assessed by high-throughput sequencing.

PubMed

Decelle, Johan; Romac, Sarah; Sasaki, Eriko; Not, Fabrice; Mahé, Frédéric

2014-01-01

Metabarcoding is a powerful tool for exploring microbial diversity in the environment, but its accurate interpretation is impeded by diverse technical (e.g. PCR and sequencing errors) and biological biases (e.g. intra-individual polymorphism) that remain poorly understood. To help interpret environmental metabarcoding datasets, we investigated the intracellular diversity of the V4 and V9 regions of the 18S rRNA gene from Acantharia and Nassellaria (radiolarians) using 454 pyrosequencing. Individual cells of radiolarians were isolated, and PCRs were performed with generalist primers to amplify the V4 and V9 regions. Different denoising procedures were employed to filter the pyrosequenced raw amplicons (Acacia, AmpliconNoise, Linkage method). For each of the six isolated cells, an average of 541 V4 and 562 V9 amplicons assigned to radiolarians were obtained, from which one numerically dominant sequence and several minor variants were found. At the 97% identity, a diversity metrics commonly used in environmental surveys, up to 5 distinct OTUs were detected in a single cell. However, most amplicons grouped within a single OTU whereas other OTUs contained very few amplicons. Different analytical methods provided evidence that most minor variants forming different OTUs correspond to PCR and sequencing artifacts. Duplicate PCR and sequencing from the same DNA extract of a single cell had only 9 to 16% of unique amplicons in common, and alignment visualization of V4 and V9 amplicons showed that most minor variants contained substitutions in highly-conserved regions. We conclude that intracellular variability of the 18S rRNA in radiolarians is very limited despite its multi-copy nature and the existence of multiple nuclei in these protists. Our study recommends some technical guidelines to conservatively discard artificial amplicons from metabarcoding datasets, and thus properly assess the diversity and richness of protists in the environment.

Pyrosequencing analysis of bacterial diversity in dental unit waterlines.

PubMed

Costa, Damien; Mercier, Anne; Gravouil, Kevin; Lesobre, Jérôme; Delafont, Vincent; Bousseau, Anne; Verdon, Julien; Imbert, Christine

2015-09-15

Some infections cases due to exposure to output water from dental unit waterlines (DUWL) have been reported in the literature. However, this type of healthcare-associated risk has remained unclear and up until now the overall bacterial composition of DUWL has been poorly documented. In this study, 454 high-throughput pyrosequencing was used to investigate the bacterial community in seven dental offices (N = 7) and to identify potential bacterial pathogenic sequences. Dental unit waters (DUW) were collected from the tap water supplying units (Incoming Water; IW) to the output exposure point of the turbine handpiece (Output water; OW) following a stagnation period (OWS), and immediately after the last patient of the sampling day (OWA). A high bacterial diversity was revealed in DUW with 394 operational taxonomic units detected at the genus level. In addition to the inter-unit variability observed, results showed increased total bacterial cell concentration and shifts in bacterial community composition and abundance at the genus level, mainly within the Gamma- and Alpha-Proteobacteria class, as water circulated in the dental unit (DU). Results showed that 96.7%, 96.8% and 97.4% of the total sequences from IW, OWS and OWA respectively were common to the 3 defined water groups, thereby highlighting a common core microbiome. Results also suggested that stagnation and DU maintenance practices were critical to composition of the bacterial community. The presence of potentially pathogenic genera was detected, including Pseudomonas and Legionella spp. Emerging and opportunistic pathogenic genera such as Mycobacterium, Propionibacterium and Stenotrophomonas were likewise recovered in DUW. For the first time, an exhaustive evaluation of the bacterial communities present in DUW was performed taking into account the circulation of water within the DU. This study highlights an ignored diversity of the DUWL bacterial community. Our findings also contribute to a better appreciation of the potential infectious risk associated with dental care and suggest the importance of better managing microbial quality in DUW. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

USDA-ARS?s Scientific Manuscript database

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

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

NASA Astrophysics Data System (ADS)

Zhu, Chenggang; Fei, Yiyan; Zhu, Xiangdong

2017-02-01

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

Functional Metagenomic Investigations of Microbial Communities in a Shallow-Sea Hydrothermal System

PubMed Central

Tang, Kai; Liu, Keshao; Jiao, Nianzhi; Zhang, Yao; Chen, Chen-Tung Arthur

2013-01-01

Little is known about the functional capability of microbial communities in shallow-sea hydrothermal systems (water depth of <200 m). This study analyzed two high-throughput pyrosequencing metagenomic datasets from the vent and the surface water in the shallow-sea hydrothermal system offshore NE Taiwan. This system exhibited distinct geochemical parameters. Metagenomic data revealed that the vent and the surface water were predominated by Epsilonproteobacteria (Nautiliales-like organisms) and Gammaproteobacteria ( Thiomicrospira -like organisms), respectively. A significant difference in microbial carbon fixation and sulfur metabolism was found between the vent and the surface water. The chemoautotrophic microorganisms in the vent and in the surface water might possess the reverse tricarboxylic acid cycle and the Calvin−Bassham−Benson cycle for carbon fixation in response to carbon dioxide highly enriched in the environment, which is possibly fueled by geochemical energy with sulfur and hydrogen. Comparative analyses of metagenomes showed that the shallow-sea metagenomes contained some genes similar to those present in other extreme environments. This study may serve as a basis for deeply understanding the genetic network and functional capability of the microbial members of shallow-sea hydrothermal systems. PMID:23940820

Transcriptome analysis of Capsicum annuum varieties Mandarin and Blackcluster: assembly, annotation and molecular marker discovery.

PubMed

Ahn, Yul-Kyun; Tripathi, Swati; Kim, Jeong-Ho; Cho, Young-Il; Lee, Hye-Eun; Kim, Do-Sun; Woo, Jong-Gyu; Cho, Myeong-Cheoul

2014-01-10

Next generation sequencing technologies have proven to be a rapid and cost-effective means to assemble and characterize gene content and identify molecular markers in various organisms. Pepper (Capsicum annuum L., Solanaceae) is a major staple vegetable crop, which is economically important and has worldwide distribution. High-throughput transcriptome profiling of two pepper cultivars, Mandarin and Blackcluster, using 454 GS-FLX pyrosequencing yielded 279,221 and 316,357 sequenced reads with a total 120.44 and 142.54Mb of sequence data (average read length of 431 and 450 nucleotides). These reads resulted from 17,525 and 16,341 'isogroups' and were assembled into 19,388 and 18,057 isotigs, and 22,217 and 13,153 singletons for both the cultivars, respectively. Assembled sequences were annotated functionally based on homology to genes in multiple public databases. Detailed sequence variant analysis identified a total of 9701 and 12,741 potential SNPs which eventually resulted in 1025 and 1059 genotype specific SNPs, for both the varieties, respectively, after examining SNP frequency distribution for each mapped unigenes. These markers for pepper will be highly valuable for marker-assisted breeding and other genetic studies. © 2013 Elsevier B.V. All rights reserved.

Characterization of gonadal transcriptomes from the turbot (Scophthalmus maximus).

PubMed

Hu, Yulong; Huang, Meng; Wang, Weiji; Guan, Jiantao; Kong, Jie

2016-01-01

The mechanisms underlying sexual reproduction and sex ratio determination remains unclear in turbot, a flatfish of great commercial value. And there is limited information in the turbot database regarding genes related to the reproductive system. Here, we conducted high-throughput transcriptome profiling of turbot gonad tissues to better understand their reproductive functions and to supply essential gene sequence information for marker-assisted selection programs in the turbot industry. In this study, two gonad libraries representing sex differences in Scophthalmus maximus yielded 453 818 high-quality reads that were assembled into 24 611 contigs and 33 713 singletons by using 454 pyrosequencing, 13 936 contigs and singletons (CS) of which were annotated using BLASTx. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses revealed that various biological functions and processes were associated with many of the annotated CS. Expression analyses showed that 510 genes were differentially expressed in males versus females; 80% of these genes were annotated. In addition, 6484 and 6036 single nucleotide polymorphisms (SNPs) were identified in male and female libraries, respectively. This transcriptome resource will serve as the foundation for cDNA or SNP microarray construction, gene expression characterization, and sex-specific linkage mapping in turbot.

Combinatorial chemoenzymatic synthesis and high-throughput screening of sialosides.

PubMed

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

2008-09-19

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

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

PubMed

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.

High-throughput sequencing reveals the incomplete, short-term recovery of infant gut microbiota following parenteral antibiotic treatment with ampicillin and gentamicin.

PubMed

Fouhy, Fiona; Guinane, Caitriona M; Hussey, Seamus; Wall, Rebecca; Ryan, C Anthony; Dempsey, Eugene M; Murphy, Brendan; Ross, R Paul; Fitzgerald, Gerald F; Stanton, Catherine; Cotter, Paul D

2012-11-01

The infant gut microbiota undergoes dramatic changes during the first 2 years of life. The acquisition and development of this population can be influenced by numerous factors, and antibiotic treatment has been suggested as one of the most significant. Despite this, however, there have been relatively few studies which have investigated the short-term recovery of the infant gut microbiota following antibiotic treatment. The aim of this study was to use high-throughput sequencing (employing both 16S rRNA and rpoB-specific primers) and quantitative PCR to compare the gut microbiota of nine infants who underwent parenteral antibiotic treatment with ampicillin and gentamicin (within 48 h of birth), 4 and 8 weeks after the conclusion of treatment, relative to that of nine matched healthy controls. The investigation revealed that the gut microbiota of the antibiotic-treated infants had significantly higher proportions of Proteobacteria (P = 0.0049) and significantly lower proportions of Actinobacteria (P = 0.00001) (and the associated genus Bifidobacterium [P = 0.0132]) as well as the genus Lactobacillus (P = 0.0182) than the untreated controls 4 weeks after the cessation of treatment. By week 8, the Proteobacteria levels remained significantly higher in the treated infants (P = 0.0049), but the Actinobacteria, Bifidobacterium, and Lactobacillus levels had recovered and were similar to those in the control samples. Despite this recovery of total Bifidobacterium numbers, rpoB-targeted pyrosequencing revealed that the number of different Bifidobacterium species present in the antibiotic-treated infants was reduced. It is thus apparent that the combined use of ampicillin and gentamicin in early life can have significant effects on the evolution of the infant gut microbiota, the long-term health implications of which remain unknown.

Influence of hydraulic regimes on bacterial community structure and composition in an experimental drinking water distribution system.

PubMed

Douterelo, I; Sharpe, R L; Boxall, J B

2013-02-01

Microbial biofilms formed on the inner-pipe surfaces of drinking water distribution systems (DWDS) can alter drinking water quality, particularly if they are mechanically detached from the pipe wall to the bulk water, such as due to changes in hydraulic conditions. Results are presented here from applying 454 pyrosequencing of the 16S ribosomal RNA (rRNA) gene to investigate the influence of different hydrological regimes on bacterial community structure and to study the potential mobilisation of material from the pipe walls to the network using a full scale, temperature-controlled experimental pipeline facility accurately representative of live DWDS. Analysis of pyrosequencing and water physico-chemical data showed that habitat type (water vs. biofilm) and hydraulic conditions influenced bacterial community structure and composition in our experimental DWDS. Bacterial community composition clearly differed between biofilms and bulk water samples. Gammaproteobacteria and Betaproteobacteria were the most abundant phyla in biofilms while Alphaproteobacteria was predominant in bulk water samples. This suggests that bacteria inhabiting biofilms, predominantly species belonging to genera Pseudomonas, Zooglea and Janthinobacterium, have an enhanced ability to express extracellular polymeric substances to adhere to surfaces and to favour co-aggregation between cells than those found in the bulk water. Highest species richness and diversity were detected in 28 days old biofilms with this being accentuated at highly varied flow conditions. Flushing altered the pipe-wall bacterial community structure but did not completely remove bacteria from the pipe walls, particularly under highly varied flow conditions, suggesting that under these conditions more compact biofilms were generated. This research brings new knowledge regarding the influence of different hydraulic regimes on the composition and structure of bacterial communities within DWDS and the implication that this might have on drinking water quality. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

PubMed

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.

Assessment of the microbial diversity of Brazilian kefir grains by PCR-DGGE and pyrosequencing analysis.

PubMed

Leite, A M O; Mayo, B; Rachid, C T C C; Peixoto, R S; Silva, J T; Paschoalin, V M F; Delgado, S

2012-09-01

The microbial diversity and community structure of three different kefir grains from different parts of Brazil were examined via the combination of two culture-independent methods: PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing. PCR-DGGE showed Lactobacillus kefiranofaciens and Lactobacillus kefiri to be the major bacterial populations in all three grains. The yeast community was dominated by Saccharomyces cerevisiae. Pyrosequencing produced a total of 14,314 partial 16S rDNA sequence reads from the three grains. Sequence analysis grouped the reads into three phyla, of which Firmicutes was dominant. Members of the genus Lactobacillus were the most abundant operational taxonomic units (OTUs) in all samples, accounting for up to 96% of the sequences. OTUs belonging to other lactic and acetic acid bacteria genera, such as Lactococcus, Leuconostoc, Streptococcus and Acetobacter, were also identified at low levels. Two of the grains showed identical DGGE profiles and a similar number of OTUs, while the third sample showed the highest diversity by both techniques. Pyrosequencing allowed the identification of bacteria that were present in small numbers and rarely associated with the microbial community of this complex ecosystem. Copyright © 2012 Elsevier Ltd. All rights reserved.

A high-throughput approach to profile RNA structure.

PubMed

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

2017-03-17

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

Clinical Neuropathology practice news 1-2014: Pyrosequencing meets clinical and analytical performance criteria for routine testing of MGMT promoter methylation status in glioblastoma

PubMed Central

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

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

DOE PAGES

Rames, Matthew; Yu, Yadong; Ren, Gang

2014-08-15

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

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

PubMed

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

2016-01-01

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

Responses of Aquatic Bacteria to Terrestrial Runoff: Effects on Community Structure and Key Taxonomic Groups

PubMed Central

Le, Huong T.; Ho, Cuong T.; Trinh, Quan H.; Trinh, Duc A.; Luu, Minh T. N.; Tran, Hai S.; Orange, Didier; Janeau, Jean L.; Merroune, Asmaa; Rochelle-Newall, Emma; Pommier, Thomas

2016-01-01

Organic fertilizer application is often touted as an economical and effective method to increase soil fertility. However, this amendment may increase dissolved organic carbon (DOC) runoff into downstream aquatic ecosystems and may consequently alter aquatic microbial community. We focused on understanding the effects of DOC runoff from soils amended with compost, vermicompost, or biochar on the aquatic microbial community of a tropical reservoir. Runoff collected from a series of rainfall simulations on soils amended with different organic fertilizers was incubated for 16 days in a series of 200 L mesocosms filled with water from a downstream reservoir. We applied 454 high throughput pyrosequencing for bacterial 16S rRNA genes to analyze microbial communities. After 16 days of incubation, the richness and evenness of the microbial communities present decreased in the mesocosms amended with any organic fertilizers, except for the evenness in the mesocosms amended with compost runoff. In contrast, they increased in the reservoir water control and soil-only amended mesocosms. Community structure was mainly affected by pH and DOC concentration. Compared to the autochthonous organic carbon produced during primary production, the addition of allochthonous DOC from these organic amendments seemed to exert a stronger effect on the communities over the period of incubation. While the Proteobacteria and Actinobacteria classes were positively associated with higher DOC concentration, the number of sequences representing key bacterial groups differed between mesocosms particularly between the biochar runoff addition and the compost or vermi-compost runoff additions. The genera of Propionibacterium spp. and Methylobacterium spp. were highly abundant in the compost runoff additions suggesting that they may represent sentinel species of complex organic carbon inputs. Overall, this work further underlines the importance of studying the off-site impacts of organic fertilizers as their impact on downstream aquatic systems is not negligible. PMID:27379034

Responses of Aquatic Bacteria to Terrestrial Runoff: Effects on Community Structure and Key Taxonomic Groups.

PubMed

Le, Huong T; Ho, Cuong T; Trinh, Quan H; Trinh, Duc A; Luu, Minh T N; Tran, Hai S; Orange, Didier; Janeau, Jean L; Merroune, Asmaa; Rochelle-Newall, Emma; Pommier, Thomas

2016-01-01

Organic fertilizer application is often touted as an economical and effective method to increase soil fertility. However, this amendment may increase dissolved organic carbon (DOC) runoff into downstream aquatic ecosystems and may consequently alter aquatic microbial community. We focused on understanding the effects of DOC runoff from soils amended with compost, vermicompost, or biochar on the aquatic microbial community of a tropical reservoir. Runoff collected from a series of rainfall simulations on soils amended with different organic fertilizers was incubated for 16 days in a series of 200 L mesocosms filled with water from a downstream reservoir. We applied 454 high throughput pyrosequencing for bacterial 16S rRNA genes to analyze microbial communities. After 16 days of incubation, the richness and evenness of the microbial communities present decreased in the mesocosms amended with any organic fertilizers, except for the evenness in the mesocosms amended with compost runoff. In contrast, they increased in the reservoir water control and soil-only amended mesocosms. Community structure was mainly affected by pH and DOC concentration. Compared to the autochthonous organic carbon produced during primary production, the addition of allochthonous DOC from these organic amendments seemed to exert a stronger effect on the communities over the period of incubation. While the Proteobacteria and Actinobacteria classes were positively associated with higher DOC concentration, the number of sequences representing key bacterial groups differed between mesocosms particularly between the biochar runoff addition and the compost or vermi-compost runoff additions. The genera of Propionibacterium spp. and Methylobacterium spp. were highly abundant in the compost runoff additions suggesting that they may represent sentinel species of complex organic carbon inputs. Overall, this work further underlines the importance of studying the off-site impacts of organic fertilizers as their impact on downstream aquatic systems is not negligible.

High-throughput determination of RNA structure by proximity ligation.

PubMed

Ramani, Vijay; Qiu, Ruolan; Shendure, Jay

2015-09-01

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

Isolation of 18 microsatellite loci in the desert mistletoe Phoradendron californicum (Santalaceae) via 454 pyrosequencing1

PubMed Central

Arroyo, Juan M.; Munguia-Vega, Adrian; Rodríguez-Estrella, Ricardo; Bascompte, Jordi

2013-01-01

• 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. PMID:25202503

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

PubMed

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

2001-07-01

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

Endophyte Microbiome Diversity in Micropropagated Atriplex canescens and Atriplex torreyi var griffithsii

PubMed Central

Lucero, Mary E.; Unc, Adrian; Cooke, Peter; Dowd, Scot; Sun, Shulei

2011-01-01

Microbial diversity associated with micropropagated Atriplex species was assessed using microscopy, isolate culturing, and sequencing. Light, electron, and confocal microscopy revealed microbial cells in aseptically regenerated leaves and roots. Clone libraries and tag-encoded FLX amplicon pyrosequencing (TEFAP) analysis amplified sequences from callus homologous to diverse fungal and bacterial taxa. Culturing isolated some seed borne endophyte taxa which could be readily propagated apart from the host. Microbial cells were observed within biofilm-like residues associated with plant cell surfaces and intercellular spaces. Various universal primers amplified both plant and microbial sequences, with different primers revealing different patterns of fungal diversity. Bacterial and fungal TEFAP followed by alignment with sequences from curated databases revealed 7 bacterial and 17 ascomycete taxa in A. canescens, and 5 bacterial taxa in A. torreyi. Additional diversity was observed among isolates and clone libraries. Micropropagated Atriplex retains a complex, intimately associated microbiome which includes diverse strains well poised to interact in manners that influence host physiology. Microbiome analysis was facilitated by high throughput sequencing methods, but primer biases continue to limit recovery of diverse sequences from even moderately complex communities. PMID:21437280

Effect of pig manure on the chemical composition and microbial diversity during co-composting with spent mushroom substrate and rice husks.

PubMed

Meng, Xingyao; Liu, Bin; Xi, Chen; Luo, Xiaosha; Yuan, Xufeng; Wang, Xiaofen; Zhu, Wanbin; Wang, Hongliang; Cui, Zongjun

2018-03-01

In this study, the impact of pig manure on the maturity of compost consisting of spent mushroom substrate and rice husks was accessed. The results showed that the addition of pig manure (SMS-PM) reached 50°C 5days earlier and lasted 15days longer than without pig manure (SMS). Furthermore, the addition of pig manure improved nutrition and germination index. High-throughput 16S rRNA pyrosequencing was used to evaluate the bacterial and fungal composition during the composting process of SMS-PM compared to SMS alone. The SMS treatment showed a relatively higher abundance of carbon-degrading microbes (Bacillaceae and Thermomyces) and plant pathogenic fungi (Sordariomycetes_unclassified) at the end of the compost. In contrast, the SMS-PM showed an increased bacterial diversity with anti-pathogen (Pseudomonas). The results indicated that the addition of pig manure improved the decomposition of refractory carbon from the spent mushroom substrate and promoted the maturity and nutritional content of the compost product. Copyright © 2017. Published by Elsevier Ltd.

Metagenomic analysis of bacterial community composition and antibiotic resistance genes in a wastewater treatment plant and its receiving surface water.

PubMed

Tang, Junying; Bu, Yuanqing; Zhang, Xu-Xiang; Huang, Kailong; He, Xiwei; Ye, Lin; Shan, Zhengjun; Ren, Hongqiang

2016-10-01

The presence of pathogenic bacteria and the dissemination of antibiotic resistance genes (ARGs) may pose big risks to the rivers that receive the effluent from municipal wastewater treatment plants (WWTPs). In this study, we investigated the changes of bacterial community and ARGs along treatment processes of one WWTP, and examined the effects of the effluent discharge on the bacterial community and ARGs in the receiving river. Pyrosequencing was applied to reveal bacterial community composition including potential bacterial pathogen, and Illumina high-throughput sequencing was used for profiling ARGs. The results showed that the WWTP had good removal efficiency on potential pathogenic bacteria (especially Arcobacter butzleri) and ARGs. Moreover, the bacterial communities of downstream and upstream of the river showed no significant difference. However, the increase in the abundance of potential pathogens and ARGs at effluent outfall was observed, indicating that WWTP effluent might contribute to the dissemination of potential pathogenic bacteria and ARGs in the receiving river. Copyright © 2016 Elsevier Inc. All rights reserved.

Stimulating short-chain fatty acids production from waste activated sludge by nano zero-valent iron.

PubMed

Luo, Jingyang; Feng, Leiyu; Chen, Yinguang; Li, Xiang; Chen, Hong; Xiao, Naidong; Wang, Dongbo

2014-10-10

An efficient and green strategy, i.e. adding nano zero-valent iron into anaerobic fermentation systems to remarkably stimulate the accumulation of short-chain fatty acids from waste activated sludge via accelerating the solubilization and hydrolysis processes has been developed. In the presence of nano zero-valent iron, not only the short-chain fatty acids production was significantly improved, but also the fermentation time for maximal short-chain fatty acids was shortened compared with those in the absence of nano zero-valent iron. Mechanism investigations showed that the solubilization of sludge, hydrolysis of solubilized substances and acidification of hydrolyzed products were all enhanced by addition of nano zero-valent iron. Also, the general microbial activity of anaerobes and relative activities of key enzymes with hydrolysis and acidification of organic matters were improved than those in the control. 454 high-throughput pyrosequencing analysis suggested that the abundance of bacteria responsible for waste activated sludge hydrolysis and short-chain fatty acids production was greatly enhanced due to nano zero-valent iron addition. Copyright © 2014 Elsevier B.V. All rights reserved.

Selective simplification and reinforcement of microbial community in autothermal thermophilic aerobic digestion to enhancing stabilization process of sewage sludge by conditioning with ferric nitrate.

PubMed

Jin, Ningben; Shou, Zongqi; Yuan, Haiping; Lou, Ziyang; Zhu, Nanwen

2016-03-01

The effect of ferric nitrate on microbial community and enhancement of stabilization process for sewage sludge was investigated in autothermal thermophilic aerobic digestion. The disinhibition of volatile fatty acids (VFA) was obtained with alteration of individual VFA concentration order. Bacterial taxonomic identification by 454 high-throughput pyrosequencing found the dominant phylum Proteobacteria in non-dosing group was converted to phylum Firmicutes in dosing group after ferric nitrate added and simplification of bacteria phylotypes was achieved. The preponderant Tepidiphilus sp. vanished, and Symbiobacterium sp. and Tepidimicrobium sp. were the most advantageous phylotypes with conditioning of ferric nitrate. Consequently, biodegradable substances in dissolved organic matters increased, which contributed to the favorable environment for microbial metabolism and resulted in acceleration of sludge stabilization. Ultimately, higher stabilization level was achieved as ratio of soluble chemical oxygen demand to total chemical oxygen demand (TCOD) decreased while TCOD reduced as well in dosing group comparing to non-dosing group. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effects of various organic carbon sources on simultaneous V(V) reduction and bioelectricity generation in single chamber microbial fuel cells.

PubMed

Hao, Liting; Zhang, Baogang; Cheng, Ming; Feng, Chuanping

2016-02-01

Four ordinary carbon sources affecting V(V) reduction and bioelectricity generation in single chamber microbial fuel cells (MFCs) were investigated. Acetate supported highest maximum power density of 589.1mW/m(2), with highest V(V) removal efficiency of 77.6% during 12h operation, compared with glucose, citrate and soluble starch. Exorbitant initial V(V) concentration led to lower V(V) removal efficiencies and power outputs. Extra addition of organics had little effect on the improvement of MFCs performance. V(V) reduction and bioelectricity generation were enhanced and then suppressed by the increase of conductivity. The larger the external resistance, the higher the V(V) removal efficiencies and voltage outputs. High-throughput 16S rRNA gene pyrosequencing analysis implied the accumulation of Enterobacter which had the capabilities of V(V) reduction, electrochemical activity and fermentation, accompanied with other functional species as Pseudomonas, Spirochaeta, Sedimentibacter and Dysgonomonas. This study steps forward to remediate V(V) contaminated environment based on MFC technology. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

DOE PAGES

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

2017-03-06

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

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

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

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

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

Are Longitudinal Patterns of Bacterial Community Composition and Dissolved Organic Matter Composition Linked Across a River Continuum? (Invited)

NASA Astrophysics Data System (ADS)

Mosher, J.; Kaplan, L. A.; Kan, J.; Findlay, R. H.; Podgorski, D. C.; McKenna, A. M.; Branan, T. L.; Griffith, C.

2013-12-01

The River Continuum Concept (RCC), an early meta-ecosystem idea, was developed without the benefit of new frontiers in molecular microbial ecology and ultra-high resolution mass spectrometry. We have applied technical advances in these areas to address a hypothesis implicit in the RCC that the upstream legacy of DOM processing contributes to the structure and function of downstream bacterial communities. DOM molecular structure and microbial community structure were measured across river networks within three distinct forested catchments. High-throughput pyrosequencing of bacterial 16S rRNA amplicons and phospholipid fatty acid analysis were used to characterize bacterial communities, and ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry characterized the molecular composition of stream water DOM. Total microbial biomass varied among river networks but showed a trend of decreasing biomass in sediment with increasing stream order. There were distinct shifts in bacterial community structure and a trend of decreasing richness was observed traveling downstream in both sediment and epilithic habitats. The bacterial richness in the first order stream sediment habitats was 7728 genera which decreased to 6597 genera in the second order sites and 4867 genera in the third order streams. The richness in the epilithic biofilm habitats was 2830 genera in the first order, 2322 genera in the second order and 1629 genera in the third order sites. Over 45% of the sediment biofilm genera and 37% of the epilithic genera were found in all three orders. In addition to shifts in bacterial richness, we observed a longitudinal shift in bacterial functional-types. In the sediment biofilms, Rhodoplanes spp. (containing rhodopsin pigment) and Bradyrhizobium spp. (nitrogen fixing bacteria) were predominately found in the heavily forested first order streams, while the cyanobacteria Limnothrix spp. was dominant in the second order streams. The third order streams had higher abundances of Sphingomonadaceae spp. and Nordella spp. (both Alphaproteobacteria). The cyanobacteria Chamaesiphon spp. was observed in highest abundance in the first and second order streams of the rock biofilm samples and the cyanobacteria Oscillatoria spp. was in highest abundance in the third order streams. Stream water samples from all orders had high lignin/tannin content and were enriched with carboxylic-rich alicyclic molecules (CRAM). There was an observable shift in in the molecular weight and relative abundance of the CRAM molecules with the CRAM molecules becoming less abundant and having lower molecular weight following the downstream gradient. Multivariate statistical analyses correlated the longitudinal patterns of changes in bacterial community structure to the DOM molecular structure and geochemical parameters across the river continuum.

The complete chloroplast genome sequence of date palm (Phoenix dactylifera L.).

PubMed

Yang, Meng; Zhang, Xiaowei; Liu, Guiming; Yin, Yuxin; Chen, Kaifu; Yun, Quanzheng; Zhao, Duojun; Al-Mssallem, Ibrahim S; Yu, Jun

2010-09-15

Date palm (Phoenix dactylifera L.), a member of Arecaceae family, is one of the three major economically important woody palms--the two other palms being oil palm and coconut tree--and its fruit is a staple food among Middle East and North African nations, as well as many other tropical and subtropical regions. Here we report a complete sequence of the data palm chloroplast (cp) genome based on pyrosequencing. After extracting 369,022 cp sequencing reads from our whole-genome-shotgun data, we put together an assembly and validated it with intensive PCR-based verification, coupled with PCR product sequencing. The date palm cp genome is 158,462 bp in length and has a typical quadripartite structure of the large (LSC, 86,198 bp) and small single-copy (SSC, 17,712 bp) regions separated by a pair of inverted repeats (IRs, 27,276 bp). Similar to what has been found among most angiosperms, the date palm cp genome harbors 112 unique genes and 19 duplicated fragments in the IR regions. The junctions between LSC/IRs and SSC/IRs show different features of sequence expansion in evolution. We identified 78 SNPs as major intravarietal polymorphisms within the population of a specific cp genome, most of which were located in genes with vital functions. Based on RNA-sequencing data, we also found 18 polycistronic transcription units and three highly expression-biased genes--atpF, trnA-UGC, and rrn23. Unlike most monocots, date palm has a typical cp genome similar to that of tobacco--with little rearrangement and gene loss or gain. High-throughput sequencing technology facilitates the identification of intravarietal variations in cp genomes among different cultivars. Moreover, transcriptomic analysis of cp genes provides clues for uncovering regulatory mechanisms of transcription and translation in chloroplasts.

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

PubMed Central

Bruni, Renato

2014-01-01

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

SNP-based real-time pyrosequencing as a sensitive and specific tool for identification and differentiation of Rickettsia species in Ixodes ricinus ticks.

PubMed

Janecek, Elisabeth; Streichan, Sabine; Strube, Christina

2012-10-18

Rickettsioses are caused by pathogenic species of the genus Rickettsia and play an important role as emerging diseases. The bacteria are transmitted to mammal hosts including humans by arthropod vectors. Since detection, especially in tick vectors, is usually based on PCR with genus-specific primers to include different occurring Rickettsia species, subsequent species identification is mainly achieved by Sanger sequencing. In the present study a real-time pyrosequencing approach was established with the objective to differentiate between species occurring in German Ixodes ticks, which are R. helvetica, R. monacensis, R. massiliae, and R. felis. Tick material from a quantitative real-time PCR (qPCR) based study on Rickettsia-infections in I. ricinus allowed direct comparison of both sequencing techniques, Sanger and real-time pyrosequencing. A sequence stretch of rickettsial citrate synthase (gltA) gene was identified to contain divergent single nucleotide polymorphism (SNP) sites suitable for Rickettsia species differentiation. Positive control plasmids inserting the respective target sequence of each Rickettsia species of interest were constructed for initial establishment of the real-time pyrosequencing approach using Qiagen's PSQ 96MA Pyrosequencing System operating in a 96-well format. The approach included an initial amplification reaction followed by the actual pyrosequencing, which is traceable by pyrograms in real-time. Afterwards, real-time pyrosequencing was applied to 263 Ixodes tick samples already detected Rickettsia-positive in previous qPCR experiments. Establishment of real-time pyrosequencing using positive control plasmids resulted in accurate detection of all SNPs in all included Rickettsia species. The method was then applied to 263 Rickettsia-positive Ixodes ricinus samples, of which 153 (58.2%) could be identified for their species (151 R. helvetica and 2 R. monacensis) by previous custom Sanger sequencing. Real-time pyrosequencing identified all Sanger-determined ticks as well as 35 previously undifferentiated ticks resulting in a total number of 188 (71.5%) identified samples. Pyrosequencing sensitivity was found to be strongly dependent on gltA copy numbers in the reaction setup. Whereas less than 101 copies in the initial amplification reaction resulted in identification of 15.1% of the samples only, the percentage increased to 54.2% at 101-102 copies, to 95.6% at >102-103 copies and reached 100% samples identified for their Rickettsia species if more than 103 copies were present in the template. The established real-time pyrosequencing approach represents a reliable method for detection and differentiation of Rickettsia spp. present in I. ricinus diagnostic material and prevalence studies. Furthermore, the method proved to be faster, more cost-effective as well as more sensitive than custom Sanger sequencing with simultaneous high specificity.

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

PubMed

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

2015-07-08

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

Low-dose fixed-target serial synchrotron crystallography.

PubMed

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

2017-04-01

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

Secondary successional trajectories of structural and catabolic bacterial communities in oil-polluted soil planted with hybrid poplar.

PubMed

Mukherjee, Shinjini; Sipilä, Timo; Pulkkinen, Pertti; Yrjälä, Kim

2015-02-01

Poplars have widely been used for rhizoremediation of a broad range of organic contaminants for the past two decades. Still, there is a knowledge gap regarding the rhizosphere-associated bacterial communities of poplars and their dynamics during the remediation process. It is envisaged that a detailed understanding of rhizosphere-associated microbial populations will greatly contribute to a better design and implementation of rhizoremediation. To investigate the long-term succession of structural and catabolic bacterial communities in oil-polluted soil planted with hybrid poplar, we carried out a 2-year field study. Hybrid aspen (Populus tremula × Populus tremuloides) seedlings were planted in polluted soil excavated from an accidental oil-spill site. Vegetated and un-vegetated soil samples were collected for microbial community analyses at seven different time points during the course of 2 years and sampling time points were chosen to cover the seasonal variation in the boreal climate zone. Bacterial community structure was accessed by means of 16S rRNA gene amplicon pyrosequencing, whereas catabolic diversity was monitored by pyrosequencing of alkane hydroxylase and extradiol dioxygenase genes. We observed a clear succession of bacterial communities on both structural and functional levels from early to late-phase communities. Sphingomonas type extradiol dioxygenases and alkane hydroxylase homologs of Rhodococcus clearly dominated the early-phase communities. The high-dominance/low-diversity functional gene communities underwent a transition to low-dominance/high-diversity communities in the late phase. These results pointed towards increased catabolic capacities and a change from specialist to generalist strategy of bacterial communities during the course of secondary succession. © 2014 John Wiley & Sons Ltd.

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

DOE PAGES

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

2016-09-23

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

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

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

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

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

Rapid detection method for Bacillus anthracis using a combination of multiplexed real-time PCR and pyrosequencing and its application for food biodefense.

PubMed

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

2015-02-01

Bacillus anthracis, the causative agent of anthrax, has the capacity to form highly resilient spores as part of its life cycle. The potential for the dissemination of these spores using food as a vehicle is a huge public health concern and, hence, requires the development of a foodborne bioterrorism response approach. In this work, we address a critical gap in food biodefense by presenting a novel, combined, sequential method involving the use of real-time PCR and pyrosequencing for the rapid, specific detection of B. anthracis spores in three food matrices: milk, apple juice, and bottled water. The food samples were experimentally inoculated with 40 CFU ml(-1), and DNA was extracted from the spores and analyzed after immunomagnetic separation. Applying the combination of multiplex real-time PCR and pyrosequencing, we successfully detected the presence of targets on both of the virulence plasmids and the chromosome. The results showed that DNA amplicons generated from a five-target multiplexed real-time PCR detection using biotin-labeled primers can be used for single-plex pyrosequencing detection. The combined use of multiplexed real-time PCR and pyrosequencing is a novel, rapid detection method for B. anthracis from food and provides a tool for accurate, quantitative identification with potential biodefense applications.

Comparative microbial diversity analyses of modern marine thrombolitic mats by barcoded pyrosequencing.

PubMed

Mobberley, Jennifer M; Ortega, Maya C; Foster, Jamie S

2012-01-01

Thrombolites are unlaminated carbonate structures that form as a result of the metabolic interactions of complex microbial mat communities. Thrombolites have a long geological history; however, little is known regarding the microbes associated with modern structures. In this study, we use a barcoded 16S rRNA gene-pyrosequencing approach coupled with morphological analysis to assess the bacterial, cyanobacterial and archaeal diversity associated with actively forming thrombolites found in Highborne Cay, Bahamas. Analyses revealed four distinct microbial mat communities referred to as black, beige, pink and button mats on the surfaces of the thrombolites. At a coarse phylogenetic resolution, the domain bacterial sequence libraries from the four mats were similar, with Proteobacteria and Cyanobacteria being the most abundant. At the finer resolution of the rRNA gene sequences, significant differences in community structure were observed, with dramatically different cyanobacterial communities. Of the four mat types, the button mats contained the highest diversity of Cyanobacteria, and were dominated by two sequence clusters with high similarity to the genus Dichothrix, an organism associated with the deposition of carbonate. Archaeal diversity was low, but varied in all mat types, and the archaeal community was predominately composed of members of the Thaumarchaeota and Euryarchaeota. The morphological and genetic data support the hypothesis that the four mat types are distinctive thrombolitic mat communities. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

Comparative metagenomic analysis of microcosm structures and lignocellulolytic enzyme systems of symbiotic biomass-degrading consortia.

PubMed

Wongwilaiwalin, Sarunyou; Laothanachareon, Thanaporn; Mhuantong, Wuttichai; Tangphatsornruang, Sithichoke; Eurwilaichitr, Lily; Igarashi, Yasuo; Champreda, Verawat

2013-10-01

Decomposition of lignocelluloses by cooperative microbial actions is an essential process of carbon cycling in nature and provides a basis for biomass conversion to fuels and chemicals in biorefineries. In this study, structurally stable symbiotic aero-tolerant lignocellulose-degrading microbial consortia were obtained from biodiversified microflora present in industrial sugarcane bagasse pile (BGC-1), cow rumen fluid (CRC-1), and pulp mill activated sludge (ASC-1) by successive subcultivation on rice straw under facultative anoxic conditions. Tagged 16S rRNA gene pyrosequencing revealed that all isolated consortia originated from highly diverse environmental microflora shared similar composite phylum profiles comprising mainly Firmicutes, reflecting convergent adaptation of microcosm structures, however, with substantial differences at refined genus level. BGC-1 comprising cellulolytic Clostridium and Acetanaerobacterium in stable coexistence with ligninolytic Ureibacillus showed the highest capability on degradation of agricultural residues and industrial pulp waste with CMCase, xylanase, and β-glucanase activities in the supernatant. Shotgun pyrosequencing of the BGC-1 metagenome indicated a markedly high relative abundance of genes encoding for glycosyl hydrolases, particularly for lignocellulytic enzymes in 26 families. The enzyme system comprised a unique composition of main-chain degrading and side-chain processing hydrolases, dominated by GH2, 3, 5, 9, 10, and 43, reflecting adaptation of enzyme profiles to the specific substrate. Gene mapping showed metabolic potential of BGC-1 for conversion of biomass sugars to various fermentation products of industrial importance. The symbiotic consortium is a promising simplified model for study of multispecies mechanisms on consolidated bioprocessing and a platform for discovering efficient synergistic enzyme systems for biotechnological application.

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

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

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

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

PubMed

Paiva, Anthony; Shou, Wilson Z

2016-08-01

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

Impact of subacute ruminal acidosis (SARA) adaptation on rumen microbiota in dairy cattle using pyrosequencing.

PubMed

Mao, S Y; Zhang, R Y; Wang, D S; Zhu, W Y

2013-12-01

The objective of this study was to evaluate the changes in bacterial populations in the rumen of dairy cattle following adaptation to subacute ruminal acidosis (SARA) using 16S rRNA gene pyrosequencing. Rumen contents were collected from four cattle adapted to either a 40% (control diet, COD) or 70% (SARA induction diet, SAID) concentrate feeds. DNA was extracted from each of the samples. Bacterial 16S rRNA genes of ruminal DNA extracts were PCR amplified with 2 bar coded primer sets and sequenced by 454 pyrosequencing. At a high taxonomic level, the percentage of Proteobacteria and Bacteroidetes were reduced by SAID feeding, whereas Firmicutes and Actinobacteria were more abundant in the SAID than in the COD group. At the genus level, as compared with the COD group, the abundances of Prevotella, Treponema, Anaeroplasma, Papillibacter, Acinetobacter and unclassified populations including unclassified Lentisphaerae, and unclassified bacteria were lower (P < 0.05), while the percentages of Ruminococcus, Atopobium, unclassified Clostridiales and Bifidobacterium were increased (P < 0.05) in the SAID group. Feeding of SAID reduced (P < 0.001) the diversity of the rumen microbial community. Taken together, our findings provide a comprehensive picture of current knowledge of the community structure of the rumen bacterial ecosystem during SARA, and enhance our understanding about the ruminal microbial ecology that may be useful in the prevention of ruminal acidosis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Searching for resistance genes to Bursaphelenchus xylophilus using high throughput screening

PubMed Central

2012-01-01

Background Pine wilt disease (PWD), caused by the pinewood nematode (PWN; Bursaphelenchus xylophilus), damages and kills pine trees and is causing serious economic damage worldwide. Although the ecological mechanism of infestation is well described, the plant’s molecular response to the pathogen is not well known. This is due mainly to the lack of genomic information and the complexity of the disease. High throughput sequencing is now an efficient approach for detecting the expression of genes in non-model organisms, thus providing valuable information in spite of the lack of the genome sequence. In an attempt to unravel genes potentially involved in the pine defense against the pathogen, we hereby report the high throughput comparative sequence analysis of infested and non-infested stems of Pinus pinaster (very susceptible to PWN) and Pinus pinea (less susceptible to PWN). Results Four cDNA libraries from infested and non-infested stems of P. pinaster and P. pinea were sequenced in a full 454 GS FLX run, producing a total of 2,083,698 reads. The putative amino acid sequences encoded by the assembled transcripts were annotated according to Gene Ontology, to assign Pinus contigs into Biological Processes, Cellular Components and Molecular Functions categories. Most of the annotated transcripts corresponded to Picea genes-25.4-39.7%, whereas a smaller percentage, matched Pinus genes, 1.8-12.8%, probably a consequence of more public genomic information available for Picea than for Pinus. The comparative transcriptome analysis showed that when P. pinaster was infested with PWN, the genes malate dehydrogenase, ABA, water deficit stress related genes and PAR1 were highly expressed, while in PWN-infested P. pinea, the highly expressed genes were ricin B-related lectin, and genes belonging to the SNARE and high mobility group families. Quantitative PCR experiments confirmed the differential gene expression between the two pine species. Conclusions Defense-related genes triggered by nematode infestation were detected in both P. pinaster and P. pinea transcriptomes utilizing 454 pyrosequencing technology. P. pinaster showed higher abundance of genes related to transcriptional regulation, terpenoid secondary metabolism (including some with nematicidal activity) and pathogen attack. P. pinea showed higher abundance of genes related to oxidative stress and higher levels of expression in general of stress responsive genes. This study provides essential information about the molecular defense mechanisms utilized by P. pinaster and P. pinea against PWN infestation and contributes to a better understanding of PWD. PMID:23134679

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

PubMed

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

2015-02-01

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

Structuring intuition with theory: The high-throughput way

NASA Astrophysics Data System (ADS)

Fornari, Marco

2015-03-01

First principles methodologies have grown in accuracy and applicability to the point where large databases can be built, shared, and analyzed with the goal of predicting novel compositions, optimizing functional properties, and discovering unexpected relationships between the data. In order to be useful to a large community of users, data should be standardized, validated, and distributed. In addition, tools to easily manage large datasets should be made available to effectively lead to materials development. Within the AFLOW consortium we have developed a simple frame to expand, validate, and mine data repositories: the MTFrame. Our minimalistic approach complement AFLOW and other existing high-throughput infrastructures and aims to integrate data generation with data analysis. We present few examples from our work on materials for energy conversion. Our intent s to pinpoint the usefulness of high-throughput methodologies to guide the discovery process by quantitatively structuring the scientific intuition. This work was supported by ONR-MURI under Contract N00014-13-1-0635 and the Duke University Center for Materials Genomics.

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

PubMed

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.

Highly sensitive detection of DNA methylation levels by using a quantum dot-based FRET method

NASA Astrophysics Data System (ADS)

Ma, Yunfei; Zhang, Honglian; Liu, Fangming; Wu, Zhenhua; Lu, Shaohua; Jin, Qinghui; Zhao, Jianlong; Zhong, Xinhua; Mao, Hongju

2015-10-01

DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR amplification for the incorporation of Alexa Fluor-647 (A647) fluorophores. DNA methylation levels can be detected qualitatively through gel analysis and quantitatively by the signal amplification from QDs to A647 during FRET. Furthermore, the methylation levels of three tumor suppressor genes, PCDHGB6, HOXA9 and RASSF1A, in 20 lung adenocarcinoma and 20 corresponding adjacent nontumorous tissue (NT) samples were measured to verify the feasibility of the QD-based FRET method and a high sensitivity for cancer detection (up to 90%) was achieved. Our QD-based FRET method is a convenient, continuous and high-throughput method, and is expected to be an alternative for detecting DNA methylation as a biomarker for certain human cancers.DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR amplification for the incorporation of Alexa Fluor-647 (A647) fluorophores. DNA methylation levels can be detected qualitatively through gel analysis and quantitatively by the signal amplification from QDs to A647 during FRET. Furthermore, the methylation levels of three tumor suppressor genes, PCDHGB6, HOXA9 and RASSF1A, in 20 lung adenocarcinoma and 20 corresponding adjacent nontumorous tissue (NT) samples were measured to verify the feasibility of the QD-based FRET method and a high sensitivity for cancer detection (up to 90%) was achieved. Our QD-based FRET method is a convenient, continuous and high-throughput method, and is expected to be an alternative for detecting DNA methylation as a biomarker for certain human cancers. Electronic supplementary information (ESI) available: Synthesis of CdSe/CdS/ZnS core/shell/shell QDs. Sequences of primers used for amplifying the promoter regions in bisulfate-modified DNA. Comparison of detected methylation levels in different gene promoters using the QD-based FRET method versus bisulfite pyrosequencing. Methylation levels of the RASSF1A gene in one pair of NT and cancer samples as indicated by pyrosequencing. Theoretical calculation of the Förster distance R0. See DOI: 10.1039/c5nr04956c

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

PubMed

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

2018-06-01

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

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

PubMed

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

2017-05-11

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

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

PubMed

Rask, Thomas S; Petersen, Bent; Chen, Donald S; Day, Karen P; Pedersen, Anders Gorm

2016-04-22

Amplicon pyrosequencing targets a known genetic region and thus inherently produces reads highly anticipated to have certain features, such as conserved nucleotide sequence, and in the case of protein coding DNA, an open reading frame. Pyrosequencing errors, consisting mainly of nucleotide insertions and deletions, are on the other hand likely to disrupt open reading frames. Such an inverse relationship between errors and expectation based on prior knowledge can be used advantageously to guide the process known as basecalling, i.e. the inference of nucleotide sequence from raw sequencing data. 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 sequencing data, particularly where extensive prior knowledge is available about the obtained sequences, for example in analysis of the immunoglobulin VDJ region where Multipass can be combined with a model for the known recombining germline genes. Multipass is available for Roche 454 data at http://www.cbs.dtu.dk/services/MultiPass-1.0 , and the concept can potentially be implemented for other sequencing technologies as well.

Introduction of the hybcell-based compact sequencing technology and comparison to state-of-the-art methodologies for KRAS mutation detection.

PubMed

Zopf, Agnes; Raim, Roman; Danzer, Martin; Niklas, Norbert; Spilka, Rita; Pröll, Johannes; Gabriel, Christian; Nechansky, Andreas; Roucka, Markus

2015-03-01

The detection of KRAS mutations in codons 12 and 13 is critical for anti-EGFR therapy strategies; however, only those methodologies with high sensitivity, specificity, and accuracy as well as the best cost and turnaround balance are suitable for routine daily testing. Here we compared the performance of compact sequencing using the novel hybcell technology with 454 next-generation sequencing (454-NGS), Sanger sequencing, and pyrosequencing, using an evaluation panel of 35 specimens. A total of 32 mutations and 10 wild-type cases were reported using 454-NGS as the reference method. Specificity ranged from 100% for Sanger sequencing to 80% for pyrosequencing. Sanger sequencing and hybcell-based compact sequencing achieved a sensitivity of 96%, whereas pyrosequencing had a sensitivity of 88%. Accuracy was 97% for Sanger sequencing, 85% for pyrosequencing, and 94% for hybcell-based compact sequencing. Quantitative results were obtained for 454-NGS and hybcell-based compact sequencing data, resulting in a significant correlation (r = 0.914). Whereas pyrosequencing and Sanger sequencing were not able to detect multiple mutated cell clones within one tumor specimen, 454-NGS and the hybcell-based compact sequencing detected multiple mutations in two specimens. Our comparison shows that the hybcell-based compact sequencing is a valuable alternative to state-of-the-art methodologies used for detection of clinically relevant point mutations.

Impact of short-chain galactooligosaccharides on the gut microbiome of lactose-intolerant individuals.

PubMed

Azcarate-Peril, M Andrea; Ritter, Andrew J; Savaiano, Dennis; Monteagudo-Mera, Andrea; Anderson, Carlton; Magness, Scott T; Klaenhammer, Todd R

2017-01-17

Directed modulation of the colonic bacteria to metabolize lactose effectively is a potentially useful approach to improve lactose digestion and tolerance. A randomized, double-blind, multisite placebo-controlled trial conducted in human subjects demonstrated that administration of a highly purified (>95%) short-chain galactooligosaccharide (GOS), designated "RP-G28," significantly improved clinical outcomes for lactose digestion and tolerance. In these individuals, stool samples were collected pretreatment (day 0), after GOS treatment (day 36), and 30 d after GOS feeding stopped and consumption of dairy products was encouraged (day 66). In this study, changes in the fecal microbiome were investigated using 16S rRNA amplicon pyrosequencing and high-throughput quantitative PCR. At day 36, bifidobacterial populations were increased in 27 of 30 of GOS subjects (90%), demonstrating a bifidogenic response in vivo. Relative abundance of lactose-fermenting Bifidobacterium, Faecalibacterium, and Lactobacillus were significantly increased in response to GOS. When dairy was introduced into the diet, lactose-fermenting Roseburia species increased from day 36 to day 66. The results indicated a definitive change in the fecal microbiome of lactose-intolerant individuals, increasing the abundance of lactose-metabolizing bacteria that were responsive to dietary adaptation to GOS. This change correlated with clinical outcomes of improved lactose tolerance.

Impact of short-chain galactooligosaccharides on the gut microbiome of lactose-intolerant individuals

PubMed Central

Azcarate-Peril, M. Andrea; Ritter, Andrew J.; Savaiano, Dennis; Monteagudo-Mera, Andrea; Anderson, Carlton; Magness, Scott T.; Klaenhammer, Todd R.

2017-01-01

Directed modulation of the colonic bacteria to metabolize lactose effectively is a potentially useful approach to improve lactose digestion and tolerance. A randomized, double-blind, multisite placebo-controlled trial conducted in human subjects demonstrated that administration of a highly purified (>95%) short-chain galactooligosaccharide (GOS), designated “RP-G28,” significantly improved clinical outcomes for lactose digestion and tolerance. In these individuals, stool samples were collected pretreatment (day 0), after GOS treatment (day 36), and 30 d after GOS feeding stopped and consumption of dairy products was encouraged (day 66). In this study, changes in the fecal microbiome were investigated using 16S rRNA amplicon pyrosequencing and high-throughput quantitative PCR. At day 36, bifidobacterial populations were increased in 27 of 30 of GOS subjects (90%), demonstrating a bifidogenic response in vivo. Relative abundance of lactose-fermenting Bifidobacterium, Faecalibacterium, and Lactobacillus were significantly increased in response to GOS. When dairy was introduced into the diet, lactose-fermenting Roseburia species increased from day 36 to day 66. The results indicated a definitive change in the fecal microbiome of lactose-intolerant individuals, increasing the abundance of lactose-metabolizing bacteria that were responsive to dietary adaptation to GOS. This change correlated with clinical outcomes of improved lactose tolerance. PMID:28049818

Treatment performance, nitrous oxide production and microbial community under low-ammonium wastewater in a CANON process.

PubMed

Mi, Weixing; Zhao, Jianqiang; Ding, Xiaoqian; Ge, Guanghuan; Zhao, Rixiang

2017-12-01

To investigate the characteristics of anaerobic ammonia oxidation for treating low-ammonium wastewater, a continuous-flow completely autotrophic nitrogen removal over nitrite (CANON) biofilm reactor was studied. At a temperature of 32 ± 1 °C and a pH between 7.5 and 8.2, two operational experiments were performed: the first one fixed the hydraulic retention time (HRT) at 10 h and gradually reduced the influent ammonium concentrations from 210 to 50 mg L -1 ; the second one fixed the influent ammonium concentration at 30 mg L -1 and gradually decreased the HRT from 10 to 3 h. The results revealed that the total nitrogen removal efficiency exceeded 80%, with a corresponding total nitrogen removal rate of 0.26 ± 0.01 kg N m -3 d -1 at the final low ammonium concentration of 30 mg L -1 . Small amounts of nitrous oxide (N 2 O) up to 0.015 ± 0.004 kg m -3 d -1 at the ammonium concentration of 210 mg L -1 were produced in the CANON process and decreased with the decrease in the influent ammonium loads. High-throughput pyrosequencing analysis indicated that the dominant functional bacteria 'Candidatus Kuenenia' under high influent ammonium levels were gradually succeeded by Armatimonadetes_gp5 under low influent ammonium levels.

High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion.

PubMed

Shiratori, Hiromi; Feinweber, Carmen; Knothe, Claudia; Lötsch, Jörn; Thomas, Dominique; Geisslinger, Gerd; Parnham, Michael J; Resch, Eduard

2016-01-01

DNA methylation is a major regulatory process of gene transcription, and aberrant DNA methylation is associated with various diseases including cancer. Many compounds have been reported to modify DNA methylation states. Despite increasing interest in the clinical application of drugs with epigenetic effects, and the use of diagnostic markers for genome-wide hypomethylation in cancer, large-scale screening systems to measure the effects of drugs on DNA methylation are limited. In this study, we improved the previously established fluorescence polarization-based global DNA methylation assay so that it is more suitable for application to human genomic DNA. Our methyl-sensitive fluorescence polarization (MSFP) assay was highly repeatable (inter-assay coefficient of variation = 1.5%) and accurate (r2 = 0.99). According to signal linearity, only 50-80 ng human genomic DNA per reaction was necessary for the 384-well format. MSFP is a simple, rapid approach as all biochemical reactions and final detection can be performed in one well in a 384-well plate without purification steps in less than 3.5 hours. Furthermore, we demonstrated a significant correlation between MSFP and the LINE-1 pyrosequencing assay, a widely used global DNA methylation assay. MSFP can be applied for the pre-screening of compounds that influence global DNA methylation states and also for the diagnosis of certain types of cancer.

Gut microbiota can transfer fiber characteristics and lipid metabolic profiles of skeletal muscle from pigs to germ-free mice.

PubMed

Yan, Honglin; Diao, Hui; Xiao, Yi; Li, Wenxia; Yu, Bing; He, Jun; Yu, Jie; Zheng, Ping; Mao, Xiangbing; Luo, Yuheng; Zeng, Benhua; Wei, Hong; Chen, Daiwen

2016-08-22

Obesity causes changes in microbiota composition, and an altered gut microbiota can transfer obesity-associated phenotypes from donors to recipients. Obese Rongchang pigs (RP) exhibited distinct fiber characteristics and lipid metabolic profiles in their muscle compared with lean Yorkshire pigs (YP). However, whether RP have a different gut microbiota than YP and whether there is a relationship between the microbiota and muscle properties are poorly understood. The present study was conducted to test whether the muscle properties can be transferred from pigs to germ-free (GF) mice. High-throughput pyrosequencing confirms the presence of distinct core microbiota between pig breeds, with alterations in taxonomic distribution and modulations in β diversity. RP displayed a significant higher Firmicutes/Bacteroidetes ratio and apparent genera differences compared with YP. Transplanting the porcine microbiota into GF mice replicated the phenotypes of the donors. RP and their GF mouse recipients exhibited a higher body fat mass, a higher slow-contracting fiber proportion, a decreased fiber size and fast IIb fiber percentage, and enhanced lipogenesis in the gastrocnemius muscle. Furthermore, the gut microbiota composition of colonized mice shared high similarity with their donor pigs. Taken together, the gut microbiota of obese pigs intrinsically influences skeletal muscle development and the lipid metabolic profiles.

High-throughput Crystallography for Structural Genomics

PubMed Central

Joachimiak, Andrzej

2009-01-01

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

Diversity, Biogeography, and Biodegradation Potential of Actinobacteria in the Deep-Sea Sediments along the Southwest Indian Ridge

PubMed Central

Chen, Ping; Zhang, Limin; Guo, Xiaoxuan; Dai, Xin; Liu, Li; Xi, Lijun; Wang, Jian; Song, Lei; Wang, Yuezhu; Zhu, Yaxin; Huang, Li; Huang, Ying

2016-01-01

The phylum Actinobacteria has been reported to be common or even abundant in deep marine sediments, however, knowledge about the diversity, distribution, and function of actinobacteria is limited. In this study, actinobacterial diversity in the deep sea along the Southwest Indian Ridge (SWIR) was investigated using both 16S rRNA gene pyrosequencing and culture-based methods. The samples were collected at depths of 1662–4000 m below water surface. Actinobacterial sequences represented 1.2–9.1% of all microbial 16S rRNA gene amplicon sequences in each sample. A total of 5 actinobacterial classes, 17 orders, 28 families, and 52 genera were detected by pyrosequencing, dominated by the classes Acidimicrobiia and Actinobacteria. Differences in actinobacterial community compositions were found among the samples. The community structure showed significant correlations to geochemical factors, notably pH, calcium, total organic carbon, total phosphorus, and total nitrogen, rather than to spatial distance at the scale of the investigation. In addition, 176 strains of the Actinobacteria class, belonging to 9 known orders, 18 families, and 29 genera, were isolated. Among these cultivated taxa, 8 orders, 13 families, and 15 genera were also recovered by pyrosequencing. At a 97% 16S rRNA gene sequence similarity, the pyrosequencing data encompassed 77.3% of the isolates but the isolates represented only 10.3% of the actinobacterial reads. Phylogenetic analysis of all the representative actinobacterial sequences and isolates indicated that at least four new orders within the phylum Actinobacteria were detected by pyrosequencing. More than half of the isolates spanning 23 genera and all samples demonstrated activity in the degradation of refractory organics, including polycyclic aromatic hydrocarbons and polysaccharides, suggesting their potential ecological functions and biotechnological applications for carbon recycling. PMID:27621725

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

PubMed

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

2017-12-21

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

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

NASA Technical Reports Server (NTRS)

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

2002-01-01

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

Comparative 454 pyrosequencing of transcripts from two olive genotypes during fruit development

PubMed Central

Alagna, Fiammetta; D'Agostino, Nunzio; Torchia, Laura; Servili, Maurizio; Rao, Rosa; Pietrella, Marco; Giuliano, Giovanni; Chiusano, Maria Luisa; Baldoni, Luciana; Perrotta, Gaetano

2009-01-01

Background Despite its primary economic importance, genomic information on olive tree is still lacking. 454 pyrosequencing was used to enrich the very few sequence data currently available for the Olea europaea species and to identify genes involved in expression of fruit quality traits. Results Fruits of Coratina, a widely cultivated variety characterized by a very high phenolic content, and Tendellone, an oleuropein-lacking natural variant, were used as starting material for monitoring the transcriptome. Four different cDNA libraries were sequenced, respectively at the beginning and at the end of drupe development. A total of 261,485 reads were obtained, for an output of about 58 Mb. Raw sequence data were processed using a four step pipeline procedure and data were stored in a relational database with a web interface. Conclusion Massively parallel sequencing of different fruit cDNA collections has provided large scale information about the structure and putative function of gene transcripts accumulated during fruit development. Comparative transcript profiling allowed the identification of differentially expressed genes with potential relevance in regulating the fruit metabolism and phenolic content during ripening. PMID:19709400

Comparison of good- and bad-quality cork: application of high-throughput sequencing of phellogenic tissue.

PubMed

Teixeira, Rita Teresa; Fortes, Ana Margarida; Pinheiro, Carla; Pereira, Helena

2014-09-01

Cork is one of the most valuable non-wood forest products and plays an important role in Mediterranean economies. The production of high-quality cork is dependent on both genome and environment, posing constraints on the industry because an ever-growing amount of bad-quality cork (BQC) development has been observed. In order to identify genes responsible for production of cork of superior quality we performed a comparative analysis using the 454 pyrosequencing approach on phellogenic tissue of good- and bad-quality samples. The transcriptional profiling showed a high number of genes differentially expressed (8.48%) from which 78.8% displayed annotation. Genes more highly represented in BQC are involved in DNA synthesis, RNA processing, proteolysis, and transcription factors related to the abiotic stress response. Putative stomatal/lenticular-associated genes which may be responsible for the disadvantageous higher number of lenticular channels in BQC are also more highly represented. BQC also showed an elevated content of free phenolics. On the other hand, good-quality cork (GQC) can be distinguished by highly expressed genes encoding heat-shock proteins. Together the results provide valuable new information about the molecular events leading to cork formation and provide putative biomarkers associated with cork quality that can be useful in breeding programmes. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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

NASA Astrophysics Data System (ADS)

Erucar, Ilknur; Keskin, Seda

2018-02-01

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

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

PubMed

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 can effectively breakdown a wide variety of complex polysaccharides.

Competitive interactions between methane- and ammonia-oxidizing bacteria modulate carbon and nitrogen cycling in paddy soil

NASA Astrophysics Data System (ADS)

Zheng, Y.; Huang, R.; Wang, B. Z.; Bodelier, P. L. E.; Jia, Z. J.

2014-06-01

Pure culture studies have demonstrated that methanotrophs and ammonia oxidizers can both carry out the oxidation of methane and ammonia. However, the expected interactions resulting from these similarities are poorly understood, especially in complex, natural environments. Using DNA-based stable isotope probing and pyrosequencing of 16S rRNA and functional genes, we report on biogeochemical and molecular evidence for growth stimulation of methanotrophic communities by ammonium fertilization, and that methane modulates nitrogen cycling by competitive inhibition of nitrifying communities in a rice paddy soil. Pairwise comparison between microcosms amended with CH4, CH4+Urea, and Urea indicated that urea fertilization stimulated methane oxidation activity 6-fold during a 19-day incubation period, while ammonia oxidation activity was significantly suppressed in the presence of CH4. Pyrosequencing of the total 16S rRNA genes revealed that urea amendment resulted in rapid growth of Methylosarcina-like MOB, and nitrifying communities appeared to be partially inhibited by methane. High-throughput sequencing of the 13C-labeled DNA further revealed that methane amendment resulted in clear growth of Methylosarcina-related MOB while methane plus urea led to an equal increase in Methylosarcina and Methylobacter-related type Ia MOB, indicating the differential growth requirements of representatives of these genera. An increase in 13C assimilation by microorganisms related to methanol oxidizers clearly indicated carbon transfer from methane oxidation to other soil microbes, which was enhanced by urea addition. The active growth of type Ia methanotrops was significantly stimulated by urea amendment, and the pronounced growth of methanol-oxidizing bacteria occurred in CH4-treated microcosms only upon urea amendment. Methane addition partially inhibited the growth of Nitrosospira and Nitrosomonas in urea-amended microcosms, as well as growth of nitrite-oxidizing bacteria. These results suggest that type I methanotrophs can outcompete type II methane oxidizers in nitrogen-rich environments, rendering the interactions among methane and ammonia oxidizers more complicated than previously appreciated.

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

NASA Astrophysics Data System (ADS)

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

2017-10-01

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

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

PubMed

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

2011-10-01

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

Multiplex pyrosequencing of InDel markers for forensic DNA analysis.

PubMed

Bus, Magdalena M; Karas, Ognjen; Allen, Marie

2016-12-01

The capillary electrophoresis (CE) technology is commonly used for fragment length separation of markers in forensic DNA analysis. In this study, pyrosequencing technology was used as an alternative and rapid tool for the analysis of biallelic InDel (insertion/deletion) markers for individual identification. The DNA typing is based on a subset of the InDel markers that are included in the Investigator ® DIPplex Kit, which are sequenced in a multiplex pyrosequencing analysis. To facilitate the analysis of degraded DNA, the polymerase chain reaction (PCR) fragments were kept short in the primer design. Samples from individuals of Swedish origin were genotyped using the pyrosequencing strategy and analysis of the Investigator ® DIPplex markers with CE. A comparison between the pyrosequencing and CE data revealed concordant results demonstrating a robust and correct genotyping by pyrosequencing. Using optimal marker combination and a directed dispensation strategy, five markers could be multiplexed and analyzed simultaneously. In this proof-of-principle study, we demonstrate that multiplex InDel pyrosequencing analysis is possible. However, further studies on degraded samples, lower DNA quantities, and mixtures will be required to fully optimize InDel analysis by pyrosequencing for forensic applications. Overall, although CE analysis is implemented in most forensic laboratories, multiplex InDel pyrosequencing offers a cost-effective alternative for some applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Deep sampling of the Palomero maize transcriptome by a high throughput strategy of pyrosequencing.

PubMed

Vega-Arreguín, Julio C; Ibarra-Laclette, Enrique; Jiménez-Moraila, Beatriz; Martínez, Octavio; Vielle-Calzada, Jean Philippe; Herrera-Estrella, Luis; Herrera-Estrella, Alfredo

2009-07-06

In-depth sequencing analysis has not been able to determine the overall complexity of transcriptional activity of a plant organ or tissue sample. In some cases, deep parallel sequencing of Expressed Sequence Tags (ESTs), although not yet optimized for the sequencing of cDNAs, has represented an efficient procedure for validating gene prediction and estimating overall gene coverage. This approach could be very valuable for complex plant genomes. In addition, little emphasis has been given to efforts aiming at an estimation of the overall transcriptional universe found in a multicellular organism at a specific developmental stage. To explore, in depth, the transcriptional diversity in an ancient maize landrace, we developed a protocol to optimize the sequencing of cDNAs and performed 4 consecutive GS20-454 pyrosequencing runs of a cDNA library obtained from 2 week-old Palomero Toluqueño maize plants. The protocol reported here allowed obtaining over 90% of informative sequences. These GS20-454 runs generated over 1.5 Million reads, representing the largest amount of sequences reported from a single plant cDNA library. A collection of 367,391 quality-filtered reads (30.09 Mb) from a single run was sufficient to identify transcripts corresponding to 34% of public maize ESTs databases; total sequences generated after 4 filtered runs increased this coverage to 50%. Comparisons of all 1.5 Million reads to the Maize Assembled Genomic Islands (MAGIs) provided evidence for the transcriptional activity of 11% of MAGIs. We estimate that 5.67% (86,069 sequences) do not align with public ESTs or annotated genes, potentially representing new maize transcripts. Following the assembly of 74.4% of the reads in 65,493 contigs, real-time PCR of selected genes confirmed a predicted correlation between the abundance of GS20-454 sequences and corresponding levels of gene expression. A protocol was developed that significantly increases the number, length and quality of cDNA reads using massive 454 parallel sequencing. We show that recurrent 454 pyrosequencing of a single cDNA sample is necessary to attain a thorough representation of the transcriptional universe present in maize, that can also be used to estimate transcript abundance of specific genes. This data suggests that the molecular and functional diversity contained in the vast native landraces remains to be explored, and that large-scale transcriptional sequencing of a presumed ancestor of the modern maize varieties represents a valuable approach to characterize the functional diversity of maize for future agricultural and evolutionary studies.

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

PubMed Central

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

2013-01-01

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

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

PubMed Central

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

2016-01-01

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

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

PubMed

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

2010-06-01

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

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

PubMed

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

2014-06-01

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

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

PubMed

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

2011-10-01

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

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

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

Structure, inter-annual recurrence, and global-scale connectivity of airborne microbial communities.

PubMed

Barberán, Albert; Henley, Jessica; Fierer, Noah; Casamayor, Emilio O

2014-07-15

Dust coming from the large deserts on Earth, such as the Sahara, can travel long distances and be dispersed over thousands of square kilometers. Remote dust deposition rates are increasing as a consequence of global change and may represent a mechanism for intercontinental microbial dispersal. Remote oligotrophic alpine lakes are particularly sensitive to dust inputs and can serve as sentinels of airborne microbial transport and the ecological consequences of accelerated intercontinental microbial migration. In this study, we applied high-throughput sequencing techniques (16S rRNA amplicon pyrosequencing) to characterize the microbial communities of atmospheric deposition collected in the Central Pyrenees (NE Spain) along three years. Additionally, bacteria from soils in Mauritania and from the air-water interface of high altitude Pyrenean lakes were also examined. Communities in aerosol deposition varied in time with a strong seasonal component of interannual similarity. Communities from the same season tended to resemble more each other than those from different seasons. Samples from disparate dates, in turn, slightly tended to have more dissimilar microbial assemblages (i.e., temporal distance decay), overall suggesting that atmospheric deposition may influence sink habitats in a temporally predictable manner. The three habitats examined (soil, deposition, and air-water interface) harbored distinct microbial communities, although airborne samples collected in the Pyrenees during Saharan dust outbreaks were closer to Mauritian soil samples than those collected during no Saharan dust episodes. The three habitats shared c.a. 1.4% of the total number of microbial sequences in the dataset. Such successful immigrants were spread in different bacterial classes. Overall, this study suggests that local and regional features may generate global trends in the dynamics and distribution of airborne microbial assemblages, and that the diversity of viable cells in the high atmosphere is likely higher than previously expected. Copyright © 2014 Elsevier B.V. All rights reserved.

Spatial and species variations in bacterial communities associated with corals from the Red Sea as revealed by pyrosequencing.

PubMed

Lee, On On; Yang, Jiangke; Bougouffa, Salim; Wang, Yong; Batang, Zenon; Tian, Renmao; Al-Suwailem, Abdulaziz; Qian, Pei-Yuan

2012-10-01

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.

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

PubMed Central

Lee, On On; Yang, Jiangke; Bougouffa, Salim; Wang, Yong; Batang, Zenon; Tian, Renmao; Al-Suwailem, Abdulaziz

2012-01-01

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. PMID:22865078

Enhanced treatment of Fischer-Tropsch wastewater using up-flow anaerobic sludge blanket system coupled with micro-electrolysis cell: A pilot scale study.

PubMed

Wang, Dexin; Han, Yuxing; Han, Hongjun; Li, Kun; Xu, Chunyan

2017-08-01

The coupling of micro-electrolysis cell (MEC) with an up-flow anaerobic sludge blanket (UASB) system in pilot scale was established for enhanced treatment of Fischer-Tropsch (F-T) wastewater. The lowest influent pH (4.99±0.10) and reduced alkali addition were accomplished under the assistance of anaerobic effluent recycling of 200% (stage 5). Simultaneously, the optimum COD removal efficiency (93.5±1.6%) and methane production (2.01±0.13m 3 /m 3 ·d) at the lower hydraulic retention time (HRT) were achieved in this stage. In addition, the dissolved iron from MEC could significantly increase the protein content of tightly bound extracellular polymeric substances (TB-EPS), which was beneficial to formation of stable granules. Furthermore, the high-throughput 16S rRNA gene pyrosequencing in this study further confirmed that Geobacter species could utilize iron oxides particles as electron conduit to perform the direct interspecies electron transfer (DIET) with Methanothrix, finally facilitating the syntrophic degradation of propionic acid and butyric acid and contributing completely methane production. Copyright © 2017 Elsevier Ltd. All rights reserved.

Sources of Bacteria in Outdoor Air across Cities in the Midwestern United States ▿ †

PubMed Central

Bowers, Robert M.; Sullivan, Amy P.; Costello, Elizabeth K.; Collett, Jeff L.; Knight, Rob; Fierer, Noah

2011-01-01

Bacteria are abundant in the atmosphere, where they often represent a major portion of the organic aerosols. Potential pathogens of plants and livestock are commonly dispersed through the atmosphere, and airborne bacteria can have important effects on human health as pathogens or triggers of allergic asthma and seasonal allergies. Despite their importance, the diversity and biogeography of airborne microorganisms remain poorly understood. We used high-throughput pyrosequencing to analyze bacterial communities present in the aerosol fraction containing fine particulate matter of ≤2.5 μm from 96 near-surface atmospheric samples collected from cities throughout the midwestern United States and found that the communities are surprisingly diverse and strongly affected by the season. We also directly compared the airborne communities to those found in hundreds of samples representing potential source environments. We show that, in addition to the more predictable sources (soils and leaf surfaces), fecal material, most likely dog feces, often represents an unexpected source of bacteria in the atmosphere at more urbanized locations during the winter. Airborne bacteria are clearly an important, but understudied, component of air quality that needs to be better integrated into efforts to measure and model pollutants in the atmosphere. PMID:21803902

Identification of microRNAs differentially expressed involved in male flower development.

PubMed

Wang, Zhengjia; Huang, Jianqin; Sun, Zhichao; Zheng, Bingsong

2015-03-01

Hickory (Carya cathayensis Sarg.) is one of the most economically important woody trees in eastern China, but its long flowering phase delays yield. Our understanding of the regulatory roles of microRNAs (miRNAs) in male flower development in hickory remains poor. Using high-throughput sequencing technology, we have pyrosequenced two small RNA libraries from two male flower differentiation stages in hickory. Analysis of the sequencing data identified 114 conserved miRNAs that belonged to 23 miRNA families, five novel miRNAs including their corresponding miRNA*s, and 22 plausible miRNA candidates. Differential expression analysis revealed 12 miRNA sequences that were upregulated in the later (reproductive) stage of male flower development. Quantitative real-time PCR showed similar expression trends as that of the deep sequencing. Novel miRNAs and plausible miRNA candidates were predicted using bioinformatic analysis methods. The miRNAs newly identified in this study have increased the number of known miRNAs in hickory, and the identification of differentially expressed miRNAs will provide new avenues for studies into miRNAs involved in the process of male flower development in hickory and other related trees.

Variation in the Gut Microbiota of Termites (Tsaitermes ampliceps) Against Different Diets.

PubMed

Su, Lijuan; Yang, Lele; Huang, Shi; Li, Yan; Su, Xiaoquan; Wang, Fengqin; Bo, Cunpei; Wang, En Tao; Song, Andong

2017-01-01

Termites are well recognized for their thriving on recalcitrant lignocellulosic diets through nutritional symbioses with gut-dwelling microbiota; however, the effects of diet changes on termite gut microbiota are poorly understood, especially for the lower termites. In this study, we employed high-throughput 454 pyrosequencing of 16S V1-V3 amplicons to compare gut microbiotas of Tsaitermes ampliceps fed with lignin-rich and lignin-poor cellulose diets after a 2-week-feeding period. As a result, the majority of bacterial taxa were shared across the treatments with different diets, but their relative abundances were modified. In particular, the relative abundance was reduced for Spirochaetes and it was increased for Proteobacteria and Bacteroides by feeding the lignin-poor diet. The evenness of gut microbiota exhibited a significant difference in response to the diet type (filter paper diets < corn stover diets < wood diets), while their richness was constant, which may be related to the lower recalcitrance of this biomass to degradation. These results have important implications for sampling and analysis strategies to probe the lignocellulose degradation features of termite gut microbiota and suggest that the dietary lignocellulose composition could cause shifting rapidly in the termite gut microbiota.

Seasonal and geographical distribution of near-surface small photosynthetic eukaryotes in the western North Pacific determined by pyrosequencing of 18S rDNA.

PubMed

Kataoka, Takafumi; Yamaguchi, Haruyo; Sato, Mayumi; Watanabe, Tsuyoshi; Taniuchi, Yukiko; Kuwata, Akira; Kawachi, Masanobu

2017-02-01

In this study, we investigated the distribution of small photosynthetic eukaryotes in the near-surface layer of the western North Pacific at four stations, including two oceanic stations where the subarctic Oyashio and subtropical Kuroshio currents influence a transition region and the bay mouth and head of the Sendai Bay, from April 2012 to May 2013. Flow cytometry was applied to sort small photosynthetic eukaryotes (<5 μm), and high-throughput sequencing of 18S rDNA was performed. Our taxonomic analysis showed that 19/195 operational taxonomic units (OTUs) were frequently distributed among all sites. Composition analysis showed that the OTUs had characteristic patterns and were divided into four main groups. Two groups reflected the low-saline water and winter season, with the characteristic OTUs belonging to diatoms; Chaetoceros and Leptocylindrus were characteristic of low saline water, and two diatom genera (Minidiscus and Minutocellus) and Cryptomonadales-related OTUs were prevalent in the winter. Our results indicate that the community composition of small photosynthetic eukaryotes seasonally changes in a dynamic manner according to variations in water properties. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Transcriptome-wide investigation of genomic imprinting in chicken.

PubMed

Frésard, Laure; Leroux, Sophie; Servin, Bertrand; Gourichon, David; Dehais, Patrice; Cristobal, Magali San; Marsaud, Nathalie; Vignoles, Florence; Bed'hom, Bertrand; Coville, Jean-Luc; Hormozdiari, Farhad; Beaumont, Catherine; Zerjal, Tatiana; Vignal, Alain; Morisson, Mireille; Lagarrigue, Sandrine; Pitel, Frédérique

2014-04-01

Genomic imprinting is an epigenetic mechanism by which alleles of some specific genes are expressed in a parent-of-origin manner. It has been observed in mammals and marsupials, but not in birds. Until now, only a few genes orthologous to mammalian imprinted ones have been analyzed in chicken and did not demonstrate any evidence of imprinting in this species. However, several published observations such as imprinted-like QTL in poultry or reciprocal effects keep the question open. Our main objective was thus to screen the entire chicken genome for parental-allele-specific differential expression on whole embryonic transcriptomes, using high-throughput sequencing. To identify the parental origin of each observed haplotype, two chicken experimental populations were used, as inbred and as genetically distant as possible. Two families were produced from two reciprocal crosses. Transcripts from 20 embryos were sequenced using NGS technology, producing ∼200 Gb of sequences. This allowed the detection of 79 potentially imprinted SNPs, through an analysis method that we validated by detecting imprinting from mouse data already published. However, out of 23 candidates tested by pyrosequencing, none could be confirmed. These results come together, without a priori, with previous statements and phylogenetic considerations assessing the absence of genomic imprinting in chicken.

High-Throughput Sequencing: A Roadmap Toward Community Ecology

PubMed Central

Poisot, Timothée; Péquin, Bérangère; Gravel, Dominique

2013-01-01

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

A high throughput spectral image microscopy system

NASA Astrophysics Data System (ADS)

Gesley, M.; Puri, R.

2018-01-01

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

Comparative analyses of fecal microbiota in Tibetan and Chinese Han living at low or high altitude by barcoded 454 pyrosequencing

PubMed Central

Li, Long; Zhao, Xin

2015-01-01

Knowledge about the impact of altitude and ethnicity on human gut microbiota is currently limited. In this study, fecal microbiota from 12 Tibetans (T group), 11 Chinese Han living in Tibet (HH group) and 12 Chinese Han living in Shaanxi province (LH group) were profiled by 454 pyrosequencing. Analysis of UniFrac principal coordinates showed significant structural changes in fecal microbiota among the three groups. There were significant differences in the composition of fecal microbiota among the three groups at phylum and genus levels. At the phylum level, the fecal samples of HH and T groups had higher relative abundances of Firmicutes, whereas the LH group had a higher relative abundance of Bacteroidetes. These changes at the phylum level reflected different dominant genus compositions. Compared with the LH group, changes of Firmicutes and Bacteroidetes were mainly due to a significant decrease of Prevotella in the HH group and were primarily attributable to significant decreases of Bacteroides and Prevotella as well as a significant increase of Catenibacterium in the T group. In conclusion, our results suggest that high altitude may contribute to shaping human gut microbiota. Genetic and dietary factors may also explain the different microbiota compositions between Tibetan and Chinese Han. PMID:26443005

Pyrosequencing analysis of free-living and attached bacterial communities in Meiliang Bay, Lake Taihu, a large eutrophic shallow lake in China.

PubMed

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.

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

PubMed

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, P<0.05). Nested 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%, P<0.01). The detection sensitivity of Sanger sequencing varied with the viral loads, especially in samples with low viral copies (HBV DNA ≤3log10 copies/ml), where the sensitivity was 78%, significantly lower than that of pyrosequencing (100%, P<0.01). Neither of the two methods yielded positive results for the negative control samples, suggesting their good specificity. Compared with nested 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.

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.

PubMed

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-MSP, pyrosequencing, and MS-HRM varied, the prognostic effect seemed similar (HR 1.74, 95 % CI 0.97-3.15; HR 1.85, 95 % CI 0.93-3.86; HR 1.83, 95 % CI 0.92-3.65, respectively). Our results show that upon optimizing and aligning four RET methylation assays with regard to primer location and sensitivity, differences in methylation frequencies and clinical sensitivities are observed; however, the effect on the marker's prognostic outcome is minimal.

Epigenetics and Epigenomics of Plants.

PubMed

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

2018-01-23

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

Determination of multiple-clone infection at allelic dimorphism site of Plasmodium vivax merozoite surface protein-1 in the Republic of Korea by pyrosequencing assay.

PubMed

Dinzouna-Boutamba, Sylvatrie-Danne; Lee, Sanghyun; Son, Ui-Han; Yun, Hae Soo; Joo, So-Young; Jeong, Sookwan; Rhee, Man Hee; Kwak, Dongmi; Xuan, Xuenan; Hong, Yeonchul; Chung, Dong-Il; Goo, Youn-Kyoung

2017-12-01

Allelic diversity leading to multiple gene polymorphisms of vivax malaria parasites has been shown to greatly contribute to antigenic variation and drug resistance, increasing the potential for multiple-clone infections within the host. Therefore, to identify multiple-clone infections and the predominant haplotype of Plasmodium vivax in a South Korean population, P. vivax merozoite surface protein-1 (PvMSP-1) was analyzed by pyrosequencing. Pyrosequencing of 156 vivax malaria-infected samples yielded 97 (62.18%) output pyrograms showing two main types of peak patterns of the dimorphic allele for threonine and alanine (T1476A). Most of the samples evaluated (88.66%) carried multiple-clone infections (wild- and mutant-types), whereas 11.34% of the same population carried only the mutant-type (1476A). In addition, each allele showed a high frequency of guanine (G) base substitution at both the first and third positions (86.07% and 81.13%, respectively) of the nucleotide combinations. Pyrosequencing of the PvMSP-1 42-kDa fragment revealed a heterogeneous parasite population, with the mutant-type dominant compared to the wild-type. Understanding the genetic diversity and multiple-clone infection rates may lead to improvements in vivax malaria prevention and strategic control plans. Further studies are needed to improve the efficacy of the pyrosequencing assay with large sample sizes and additional nucleotide positions. Copyright © 2017 Elsevier B.V. All rights reserved.

High performance hybrid magnetic structure for biotechnology applications

DOEpatents

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

2009-02-03

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

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

NASA Astrophysics Data System (ADS)

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

2018-03-01

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

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

NASA Astrophysics Data System (ADS)

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

2010-06-01

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

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

NASA Astrophysics Data System (ADS)

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

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

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

PubMed

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

2015-01-01

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

A data set from flash X-ray imaging of carboxysomes

NASA Astrophysics Data System (ADS)

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

2016-08-01

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

Microbial Biogeography of Public Restroom Surfaces

PubMed Central

Flores, Gilberto E.; Bates, Scott T.; Knights, Dan; Lauber, Christian L.; Stombaugh, Jesse; Knight, Rob; Fierer, Noah

2011-01-01

We spend the majority of our lives indoors where we are constantly exposed to bacteria residing on surfaces. However, the diversity of these surface-associated communities is largely unknown. We explored the biogeographical patterns exhibited by bacteria across ten surfaces within each of twelve public restrooms. Using high-throughput barcoded pyrosequencing of the 16 S rRNA gene, we identified 19 bacterial phyla across all surfaces. Most sequences belonged to four phyla: Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria. The communities clustered into three general categories: those found on surfaces associated with toilets, those on the restroom floor, and those found on surfaces routinely touched with hands. On toilet surfaces, gut-associated taxa were more prevalent, suggesting fecal contamination of these surfaces. Floor surfaces were the most diverse of all communities and contained several taxa commonly found in soils. Skin-associated bacteria, especially the Propionibacteriaceae, dominated surfaces routinely touched with our hands. Certain taxa were more common in female than in male restrooms as vagina-associated Lactobacillaceae were widely distributed in female restrooms, likely from urine contamination. Use of the SourceTracker algorithm confirmed many of our taxonomic observations as human skin was the primary source of bacteria on restroom surfaces. Overall, these results demonstrate that restroom surfaces host relatively diverse microbial communities dominated by human-associated bacteria with clear linkages between communities on or in different body sites and those communities found on restroom surfaces. More generally, this work is relevant to the public health field as we show that human-associated microbes are commonly found on restroom surfaces suggesting that bacterial pathogens could readily be transmitted between individuals by the touching of surfaces. Furthermore, we demonstrate that we can use high-throughput analyses of bacterial communities to determine sources of bacteria on indoor surfaces, an approach which could be used to track pathogen transmission and test the efficacy of hygiene practices. PMID:22132229

Exploring Pandora's Box: Potential and Pitfalls of Low Coverage Genome Surveys for Evolutionary Biology

PubMed Central

Leese, Florian; Mayer, Christoph; Agrawal, Shobhit; Dambach, Johannes; Dietz, Lars; Doemel, Jana S.; Goodall-Copstake, William P.; Held, Christoph; Jackson, Jennifer A.; Lampert, Kathrin P.; Linse, Katrin; Macher, Jan N.; Nolzen, Jennifer; Raupach, Michael J.; Rivera, Nicole T.; Schubart, Christoph D.; Striewski, Sebastian; Tollrian, Ralph; Sands, Chester J.

2012-01-01

High throughput sequencing technologies are revolutionizing genetic research. With this “rise of the machines”, genomic sequences can be obtained even for unknown genomes within a short time and for reasonable costs. This has enabled evolutionary biologists studying genetically unexplored species to identify molecular markers or genomic regions of interest (e.g. micro- and minisatellites, mitochondrial and nuclear genes) by sequencing only a fraction of the genome. However, when using such datasets from non-model species, it is possible that DNA from non-target contaminant species such as bacteria, viruses, fungi, or other eukaryotic organisms may complicate the interpretation of the results. In this study we analysed 14 genomic pyrosequencing libraries of aquatic non-model taxa from four major evolutionary lineages. We quantified the amount of suitable micro- and minisatellites, mitochondrial genomes, known nuclear genes and transposable elements and searched for contamination from various sources using bioinformatic approaches. Our results show that in all sequence libraries with estimated coverage of about 0.02–25%, many appropriate micro- and minisatellites, mitochondrial gene sequences and nuclear genes from different KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways could be identified and characterized. These can serve as markers for phylogenetic and population genetic analyses. A central finding of our study is that several genomic libraries suffered from different biases owing to non-target DNA or mobile elements. In particular, viruses, bacteria or eukaryote endosymbionts contributed significantly (up to 10%) to some of the libraries analysed. If not identified as such, genetic markers developed from high-throughput sequencing data for non-model organisms may bias evolutionary studies or fail completely in experimental tests. In conclusion, our study demonstrates the enormous potential of low-coverage genome survey sequences and suggests bioinformatic analysis workflows. The results also advise a more sophisticated filtering for problematic sequences and non-target genome sequences prior to developing markers. PMID:23185309

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

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

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

2009-05-26

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

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

PubMed

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

2016-05-24

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

Understanding microbial ecology can help improve biogas production in AD.

PubMed

Ferguson, Robert M W; Coulon, Frédéric; Villa, Raffaella

2018-06-16

454-Pyrosequencing and lipid fingerprinting were used to link anaerobic digestion (AD) process parameters (pH, alkalinity, volatile fatty acids (VFAs), biogas production and methane content) with the reactor microbial community structure and composition. AD microbial communities underwent stress conditions after changes in organic loading rate and digestion substrates. 454-Pyrosequencing analysis showed that, irrespectively of the substrate digested, methane content and pH were always significantly, and positively, correlated with community evenness. In AD, microbial communities with more even distributions of diversity are able to use parallel metabolic pathways and have greater functional stability; hence, they are capable of adapting and responding to disturbances. In all reactors, a decrease in methane content to <30% was always correlated with a 50% increase of Firmicutes sequences (particularly in operational taxonomic units (OTUs) related to Ruminococcaceae and Veillonellaceae). Whereas digesters producing higher methane content (above 60%), contained a high number of sequences related to Synergistetes and unidentified bacterial OTUs. Finally, lipid fingerprinting demonstrated that, under stress, the decrease in archaeal biomass was higher than the bacterial one, and that archaeal Phospholipid etherlipids (PLEL) levels were correlated to reactor performances. These results demonstrate that, across a number of parameters such as lipids, alpha and beta diversity, and OTUs, knowledge of the microbial community structure can be used to predict, monitor, or optimise AD performance. Copyright © 2018 Elsevier B.V. All rights reserved.

High throughput imaging cytometer with acoustic focussing.

PubMed

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

2015-10-31

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

Advancements in zebrafish applications for 21st century toxicology.

PubMed

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

2016-05-01

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

Advancements in zebrafish applications for 21st century toxicology

PubMed Central

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

2016-01-01

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

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

PubMed Central

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

2014-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2015-12-01

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

The JCSG high-throughput structural biology pipeline.

PubMed

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

2010-10-01

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

Optimization and high-throughput screening of antimicrobial peptides.

PubMed

Blondelle, Sylvie E; Lohner, Karl

2010-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2018-01-01

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

The use of high throughput DNA sequence analysis to assess the endophytic microbiome of date palm roots grown under different levels of salt stress.

PubMed

Yaish, Mahmoud W; Al-Harrasi, Ibtisam; Alansari, Aliya S; Al-Yahyai, Rashid; Glick, Bernard R

2016-09-01

Date palms are able to grow under diverse abiotic stress conditions including in saline soils, where microbial communities may be help in the plant's salinity tolerance. These communities able to produce specific growth promoting substances can enhance date palm growth in a saline environment. However, these communities are poorly defined. In the work reported here, the date palm endophytic bacterial and fungal communities were identified using the pyrosequencing method, and the microbial differential abundance in the root upon exposure to salinity stress was estimated. Approximately 150,061 reads were produced from the analysis of six ribosomal DNA libraries, which were prepared from endophytic microorganisms colonizing date palm root tissues. DNA sequence analysis of these libraries predicted the presence of a variety of bacterial and fungal endophytic species, some known and others unknown. The microbial community compositions of 30% and 8% of the bacterial and fungal species, respectively, were significantly (p ≤ 0.05) altered in response to salinity stress. Differential enrichment analysis showed that microbe diversity indicated by the Chao, Shannon and Simpson indices were slightly reduced, however, the overall microbial community structures were not significantly affected as a consequence of salinity. This may reflect a buffering effect by the host plant on the internal environments that these communities are colonizing. Some of the endophytes identified in this study were strains that were previously isolated from saline and marine environments. This suggests possible interactions with the plant that are favorable to salinity tolerance in date palm. [Int Microbiol 19(3):143-155 (2016)]. Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.

Deduction and Analysis of the Interacting Stress Response Pathways of Metal/Radionuclide-reducing Bacteria

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

Zhou, Jizhong; He, Zhili

2010-02-28

Project Title: Deduction and Analysis of the Interacting Stress Response Pathways of Metal/Radionuclide-reducing Bacteria DOE Grant Number: DE-FG02-06ER64205 Principal Investigator: Jizhong (Joe) Zhou (University of Oklahoma) Key members: Zhili He, Aifen Zhou, Christopher Hemme, Joy Van Nostrand, Ye Deng, and Qichao Tu Collaborators: Terry Hazen, Judy Wall, Adam Arkin, Matthew Fields, Aindrila Mukhopadhyay, and David Stahl Summary Three major objectives have been conducted in the Zhou group at the University of Oklahoma (OU): (i) understanding of gene function, regulation, network and evolution of Desulfovibrio vugaris Hildenborough in response to environmental stresses, (ii) development of metagenomics technologies for microbial community analysis,more » and (iii) functional characterization of microbial communities with metagenomic approaches. In the past a few years, we characterized four CRP/FNR regulators, sequenced ancestor and evolved D. vulgaris strains, and functionally analyzed those mutated genes identified in salt-adapted strains. Also, a new version of GeoChip 4.0 has been developed, which also includes stress response genes (StressChip), and a random matrix theory-based conceptual framework for identifying functional molecular ecological networks has been developed with the high throughput functional gene array hybridization data as well as pyrosequencing data from 16S rRNA genes. In addition, GeoChip and sequencing technologies as well as network analysis approaches have been used to analyze microbial communities from different habitats. Those studies provide a comprehensive understanding of gene function, regulation, network, and evolution in D. vulgaris, and microbial community diversity, composition and structure as well as their linkages with environmental factors and ecosystem functioning, which has resulted in more than 60 publications.« less

Rapid Molecular Identification of Pathogenic Yeasts by Pyrosequencing Analysis of 35 Nucleotides of Internal Transcribed Spacer 2 ▿

PubMed Central

Borman, Andrew M.; Linton, Christopher J.; Oliver, Debra; Palmer, Michael D.; Szekely, Adrien; Johnson, Elizabeth M.

2010-01-01

Rapid identification of yeast species isolates from clinical samples is particularly important given their innately variable antifungal susceptibility profiles. Here, we have evaluated the utility of pyrosequencing analysis of a portion of the internal transcribed spacer 2 region (ITS2) for identification of pathogenic yeasts. A total of 477 clinical isolates encompassing 43 different fungal species were subjected to pyrosequencing analysis in a strictly blinded study. The molecular identifications produced by pyrosequencing were compared with those obtained using conventional biochemical tests (AUXACOLOR2) and following PCR amplification and sequencing of the D1-D2 portion of the nuclear 28S large rRNA gene. More than 98% (469/477) of isolates encompassing 40 of the 43 fungal species tested were correctly identified by pyrosequencing of only 35 bp of ITS2. Moreover, BLAST searches of the public synchronized databases with the ITS2 pyrosequencing signature sequences revealed that there was only minimal sequence redundancy in the ITS2 under analysis. In all cases, the pyrosequencing signature sequences were unique to the yeast species (or species complex) under investigation. Finally, when pyrosequencing was combined with the Whatman FTA paper technology for the rapid extraction of fungal genomic DNA, molecular identification could be accomplished within 6 h from the time of starting from pure cultures. PMID:20702674

Neutron reflecting supermirror structure

DOEpatents

Wood, J.L.

1992-12-01

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

Neutron reflecting supermirror structure

DOEpatents

Wood, James L.

1992-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2016-09-01

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

Custom Super-Resolution Microscope for the Structural Analysis of Nanostructures

DTIC Science & Technology

2018-05-29

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

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

PubMed

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

2011-01-01

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

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

PubMed

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-Tibetan Plateau provide a valuable background for further studies on biogeochemical processes in this distinct ecosystem.

Microbial Diversity in Hummock and Hollow Soils of Three Wetlands on the Qinghai-Tibetan Plateau Revealed by 16S rRNA Pyrosequencing

PubMed Central

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-Tibetan Plateau provide a valuable background for further studies on biogeochemical processes in this distinct ecosystem. PMID:25078273

Bacterial Diversity and Community Structure of Supragingival Plaques in Adults with Dental Health or Caries Revealed by 16S Pyrosequencing

PubMed Central

Xiao, Cuicui; Ran, Shujun; Huang, Zhengwei; Liang, Jingping

2016-01-01

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. α diversity analysis showed that the microbial diversity in healthy plaques 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. Using LEfSe analysis, 25 differentially abundant taxa were identified as potential biomarkers. Key genera (27) that potentially contributed to the differential distributions of plaque microbiota between groups were identified by PLS-DA analysis. Finally, co-occurrence network analysis and function predictions were performed. Treatment strategies directed toward modulating microbial interactions and their functional output should be further developed. PMID:27499752

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

PubMed

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.

Molecular analysis of the bacterial microbiome in the forestomach fluid from the dromedary camel (Camelus dromedarius).

PubMed

Bhatt, Vaibhav D; Dande, Suchitra S; Patil, Nitin V; Joshi, Chaitanya G

2013-04-01

Rumen microorganisms play an important role in ruminant digestion and absorption of nutrients and have great potential applications in the field of rumen adjusting, food fermentation and biomass utilization etc. In order to investigate the composition of microorganisms in the rumen of camel (Camelus dromedarius), this study delves in the microbial diversity by culture-independent approach. It includes comparison of rumen samples investigated in the present study to other currently available metagenomes to reveal potential differences in rumen microbial systems. Pyrosequencing based metagenomics was applied to analyze phylogenetic and metabolic profiles by MG-RAST, a web based tool. Pyrosequencing of camel rumen sample yielded 8,979,755 nucleotides assembled to 41,905 sequence reads with an average read length of 214 nucleotides. Taxonomic analysis of metagenomic reads indicated Bacteroidetes (55.5 %), Firmicutes (22.7 %) and Proteobacteria (9.2 %) phyla as predominant camel rumen taxa. At a finer phylogenetic resolution, Bacteroides species dominated the camel rumen metagenome. Functional analysis revealed that clustering-based subsystem and carbohydrate metabolism were the most abundant SEED subsystem representing 17 and 13 % of camel metagenome, respectively. A high taxonomic and functional similarity of camel rumen was found with the cow metagenome which is not surprising given the fact that both are mammalian herbivores with similar digestive tract structures and functions. Combined pyrosequencing approach and subsystems-based annotations available in the SEED database allowed us access to understand the metabolic potential of these microbiomes. Altogether, these data suggest that agricultural and animal husbandry practices can impose significant selective pressures on the rumen microbiota regardless of rumen type. The present study provides a baseline for understanding the complexity of camel rumen microbial ecology while also highlighting striking similarities and differences when compared to other animal gastrointestinal environments.

A Multidisciplinary Approach to High Throughput Nuclear Magnetic Resonance Spectroscopy

PubMed Central

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

2016-01-01

Nuclear Magnetic Resonance (NMR) is a non-contact, powerful structure-elucidation technique for biochemical analysis. NMR spectroscopy is used extensively in a variety of life science applications including drug discovery. However, existing NMR technology is limited in that it cannot run a large number of experiments simultaneously in one unit. Recent advances in micro-fabrication technologies have attracted the attention of researchers to overcome these limitations and significantly accelerate the drug discovery process by developing the next generation of high-throughput NMR spectrometers using Complementary Metal Oxide Semiconductor (CMOS). In this paper, we examine this paradigm shift and explore new design strategies for the development of the next generation of high-throughput NMR spectrometers using CMOS technology. A CMOS NMR system consists of an array of high sensitivity micro-coils integrated with interfacing radio-frequency circuits on the same chip. Herein, we first discuss the key challenges and recent advances in the field of CMOS NMR technology, and then a new design strategy is put forward for the design and implementation of highly sensitive and high-throughput CMOS NMR spectrometers. We thereafter discuss the functionality and applicability of the proposed techniques by demonstrating the results. For microelectronic researchers starting to work in the field of CMOS NMR technology, this paper serves as a tutorial with comprehensive review of state-of-the-art technologies and their performance levels. Based on these levels, the CMOS NMR approach offers unique advantages for high resolution, time-sensitive and high-throughput bimolecular analysis required in a variety of life science applications including drug discovery. PMID:27294925

Optimal forwarding ratio on dynamical networks with heterogeneous mobility

NASA Astrophysics Data System (ADS)

Gan, Yu; Tang, Ming; Yang, Hanxin

2013-05-01

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

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

NASA Astrophysics Data System (ADS)

Buongiorno Nardelli, Marco

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

High-throughput methods for electron crystallography.

PubMed

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

2013-01-01

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

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

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

Yamada, Yusuke; Hiraki, Masahiko; Sasajima, Kumiko

2010-06-23

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

Effect of monochloramine treatment on the microbial ecology of Legionella and associated bacterial populations in a hospital hot water system.

PubMed

Baron, Julianne L; Harris, J Kirk; Holinger, Eric P; Duda, Scott; Stevens, Mark J; Robertson, Charles E; Ross, Kimberly A; Pace, Norman R; Stout, Janet E

2015-05-01

Opportunistic pathogens, including Legionella spp. and non-tuberculous mycobacteria, can thrive in building hot water systems despite municipal and traditional on-site chlorine disinfection. Monochloramine is a relatively new approach to on-site disinfection, but the microbiological impact of on-site chloramine use has not been well studied. We hypothesized that comparison of the microbial ecology associated with monochloramine treatment versus no on-site treatment would yield highly dissimilar bacterial communities. Hot water samples were collected monthly from 7 locations for three months from two buildings in a Pennsylvania hospital complex supplied with common municipal water: (1) a hospital administrative building (no on-site treatment) and (2) an adjacent acute-care hospital treated on-site with monochloramine to control Legionella spp. Water samples were subjected to DNA extraction, rRNA PCR, and 454 pyrosequencing. Stark differences in the microbiome of the chloraminated water and the control were observed. Bacteria in the treated samples were primarily Sphingomonadales and Limnohabitans, whereas Flexibacter and Planctomycetaceae predominated in untreated control samples. Serendipitously, one sampling month coincided with dysfunction of the on-site disinfection system that resulted in a Legionella bloom detected by sequencing and culture. This study also demonstrates the potential utility of high-throughput DNA sequencing to monitor microbial ecology in water systems. Copyright © 2015 Elsevier GmbH. All rights reserved.

Viral quasispecies inference from 454 pyrosequencing

PubMed Central

2013-01-01

Background Many potentially life-threatening infectious viruses are highly mutable in nature. Characterizing the fittest variants within a quasispecies from infected patients is expected to allow unprecedented opportunities to investigate the relationship between quasispecies diversity and disease epidemiology. The advent of next-generation sequencing technologies has allowed the study of virus diversity with high-throughput sequencing, although these methods come with higher rates of errors which can artificially increase diversity. Results Here we introduce a novel computational approach that incorporates base quality scores from next-generation sequencers for reconstructing viral genome sequences that simultaneously infers the number of variants within a quasispecies that are present. Comparisons on simulated and clinical data on dengue virus suggest that the novel approach provides a more accurate inference of the underlying number of variants within the quasispecies, which is vital for clinical efforts in mapping the within-host viral diversity. Sequence alignments generated by our approach are also found to exhibit lower rates of error. Conclusions The ability to infer the viral quasispecies colony that is present within a human host provides the potential for a more accurate classification of the viral phenotype. Understanding the genomics of viruses will be relevant not just to studying how to control or even eradicate these viral infectious diseases, but also in learning about the innate protection in the human host against the viruses. PMID:24308284

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

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

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

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

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

DOE PAGES

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

2015-10-07

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

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

NASA Technical Reports Server (NTRS)

Maluf, David A.; Tran, Peter B.

2003-01-01

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

Mitochondrial sequence analysis for forensic identification using pyrosequencing technology.

PubMed

Andréasson, H; Asp, A; Alderborn, A; Gyllensten, U; Allen, M

2002-01-01

Over recent years, requests for mtDNA analysis in the field of forensic medicine have notably increased, and the results of such analyses have proved to be very useful in forensic cases where nuclear DNA analysis cannot be performed. Traditionally, mtDNA has been analyzed by DNA sequencing of the two hypervariable regions, HVI and HVII, in the D-loop. DNA sequence analysis using the conventional Sanger sequencing is very robust but time consuming and labor intensive. By contrast, mtDNA analysis based on the pyrosequencing technology provides fast and accurate results from the human mtDNA present in many types of evidence materials in forensic casework. The assay has been developed to determine polymorphic sites in the mitochondrial D-loop as well as the coding region to further increase the discrimination power of mtDNA analysis. The pyrosequencing technology for analysis of mtDNA polymorphisms has been tested with regard to sensitivity, reproducibility, and success rate when applied to control samples and actual casework materials. The results show that the method is very accurate and sensitive; the results are easily interpreted and provide a high success rate on casework samples. The panel of pyrosequencing reactions for the mtDNA polymorphisms were chosen to result in an optimal discrimination power in relation to the number of bases determined.

Toxicokinetic and Dosimetry Modeling Tools for Exposure ...

EPA Pesticide Factsheets

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

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

PubMed

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

2015-10-01

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

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

EPA Science Inventory

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

Protein Structures Revealed at Record Pace

ScienceCinema

Hura, Greg

2017-12-11

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

Protein Structures Revealed at Record Pace

ScienceCinema

Greg Hura

2017-12-09

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

The Complete Chloroplast Genome Sequence of Date Palm (Phoenix dactylifera L.)

PubMed Central

Yang, Meng; Zhang, Xiaowei; Liu, Guiming; Yin, Yuxin; Chen, Kaifu; Yun, Quanzheng; Zhao, Duojun; Al-Mssallem, Ibrahim S.; Yu, Jun

2010-01-01

Background Date palm (Phoenix dactylifera L.), a member of Arecaceae family, is one of the three major economically important woody palms—the two other palms being oil palm and coconut tree—and its fruit is a staple food among Middle East and North African nations, as well as many other tropical and subtropical regions. Here we report a complete sequence of the data palm chloroplast (cp) genome based on pyrosequencing. Methodology/Principal Findings After extracting 369,022 cp sequencing reads from our whole-genome-shotgun data, we put together an assembly and validated it with intensive PCR-based verification, coupled with PCR product sequencing. The date palm cp genome is 158,462 bp in length and has a typical quadripartite structure of the large (LSC, 86,198 bp) and small single-copy (SSC, 17,712 bp) regions separated by a pair of inverted repeats (IRs, 27,276 bp). Similar to what has been found among most angiosperms, the date palm cp genome harbors 112 unique genes and 19 duplicated fragments in the IR regions. The junctions between LSC/IRs and SSC/IRs show different features of sequence expansion in evolution. We identified 78 SNPs as major intravarietal polymorphisms within the population of a specific cp genome, most of which were located in genes with vital functions. Based on RNA-sequencing data, we also found 18 polycistronic transcription units and three highly expression-biased genes—atpF, trnA-UGC, and rrn23. Conclusions Unlike most monocots, date palm has a typical cp genome similar to that of tobacco—with little rearrangement and gene loss or gain. High-throughput sequencing technology facilitates the identification of intravarietal variations in cp genomes among different cultivars. Moreover, transcriptomic analysis of cp genes provides clues for uncovering regulatory mechanisms of transcription and translation in chloroplasts. PMID:20856810

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

PubMed Central

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

2015-01-01

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

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

NASA Astrophysics Data System (ADS)

Miers, Collier Stephen

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

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

PubMed

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

2018-01-01

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

High-Throughput Characterization of Vapor-Deposited Organic Glasses

NASA Astrophysics Data System (ADS)

Dalal, Shakeel S.

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

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

PubMed Central

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

2017-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2017-03-01

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

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

PubMed Central

2011-01-01

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

Probabilistic Assessment of High-Throughput Wireless Sensor Networks

PubMed Central

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

2016-01-01

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

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

PubMed

Zhang, Aihua; Sun, Hui; Wang, Xijun

2018-05-01

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

An improved genome assembly uncovers prolific tandem repeats in Atlantic cod.

PubMed

Tørresen, Ole K; Star, Bastiaan; Jentoft, Sissel; Reinar, William B; Grove, Harald; Miller, Jason R; Walenz, Brian P; Knight, James; Ekholm, Jenny M; Peluso, Paul; Edvardsen, Rolf B; Tooming-Klunderud, Ave; Skage, Morten; Lien, Sigbjørn; Jakobsen, Kjetill S; Nederbragt, Alexander J

2017-01-18

The first Atlantic cod (Gadus morhua) genome assembly published in 2011 was one of the early genome assemblies exclusively based on high-throughput 454 pyrosequencing. Since then, rapid advances in sequencing technologies have led to a multitude of assemblies generated for complex genomes, although many of these are of a fragmented nature with a significant fraction of bases in gaps. The development of long-read sequencing and improved software now enable the generation of more contiguous genome assemblies. By combining data from Illumina, 454 and the longer PacBio sequencing technologies, as well as integrating the results of multiple assembly programs, we have created a substantially improved version of the Atlantic cod genome assembly. The sequence contiguity of this assembly is increased fifty-fold and the proportion of gap-bases has been reduced fifteen-fold. Compared to other vertebrates, the assembly contains an unusual high density of tandem repeats (TRs). Indeed, retrospective analyses reveal that gaps in the first genome assembly were largely associated with these TRs. We show that 21% of the TRs across the assembly, 19% in the promoter regions and 12% in the coding sequences are heterozygous in the sequenced individual. The inclusion of PacBio reads combined with the use of multiple assembly programs drastically improved the Atlantic cod genome assembly by successfully resolving long TRs. The high frequency of heterozygous TRs within or in the vicinity of genes in the genome indicate a considerable standing genomic variation in Atlantic cod populations, which is likely of evolutionary importance.

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

PubMed

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

2017-10-18

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

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

NASA Astrophysics Data System (ADS)

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

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

Predicting hepatotoxicity using ToxCast in vitro bioactivity and chemical structure

EPA Science Inventory

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

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

NASA Astrophysics Data System (ADS)

Rajen, Nicholas; Coh, Sinisa

2018-05-01

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

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

PubMed Central

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

2017-01-01

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

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

PubMed

Zhou, Jizhong; He, Zhili; Yang, Yunfeng; Deng, Ye; Tringe, Susannah G; Alvarez-Cohen, Lisa

2015-01-27

Understanding the structure, functions, activities and dynamics of microbial communities in natural environments is one of the grand challenges of 21st century science. To address this challenge, over the past decade, numerous technologies have been developed for interrogating microbial communities, of which some are amenable to exploratory work (e.g., high-throughput sequencing and phenotypic screening) and others depend on reference genes or genomes (e.g., phylogenetic and functional gene arrays). Here, we provide a critical review and synthesis of the most commonly applied "open-format" and "closed-format" detection technologies. We discuss their characteristics, advantages, and disadvantages within the context of environmental applications and focus on analysis of complex microbial systems, such as those in soils, in which diversity is high and reference genomes are few. In addition, we discuss crucial issues and considerations associated with applying complementary high-throughput molecular technologies to address important ecological questions. Copyright © 2015 Zhou et al.

High-Throughput Metagenomic Technologies for Complex Microbial Community Analysis: Open and Closed Formats

PubMed Central

He, Zhili; Yang, Yunfeng; Deng, Ye; Tringe, Susannah G.; Alvarez-Cohen, Lisa

2015-01-01

ABSTRACT   Understanding the structure, functions, activities and dynamics of microbial communities in natural environments is one of the grand challenges of 21st century science. To address this challenge, over the past decade, numerous technologies have been developed for interrogating microbial communities, of which some are amenable to exploratory work (e.g., high-throughput sequencing and phenotypic screening) and others depend on reference genes or genomes (e.g., phylogenetic and functional gene arrays). Here, we provide a critical review and synthesis of the most commonly applied “open-format” and “closed-format” detection technologies. We discuss their characteristics, advantages, and disadvantages within the context of environmental applications and focus on analysis of complex microbial systems, such as those in soils, in which diversity is high and reference genomes are few. In addition, we discuss crucial issues and considerations associated with applying complementary high-throughput molecular technologies to address important ecological questions. PMID:25626903

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

DOE PAGES

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

2015-01-27

Understanding the structure, functions, activities and dynamics of microbial communities in natural environments is one of the grand challenges of 21st century science. To address this challenge, over the past decade, numerous technologies have been developed for interrogating microbial communities, of which some are amenable to exploratory work (e.g., high-throughput sequencing and phenotypic screening) and others depend on reference genes or genomes (e.g., phylogenetic and functional gene arrays). Here, we provide a critical review and synthesis of the most commonly applied “open-format” and “closed-format” detection technologies. We discuss their characteristics, advantages, and disadvantages within the context of environmental applications andmore » focus on analysis of complex microbial systems, such as those in soils, in which diversity is high and reference genomes are few. In addition, we discuss crucial issues and considerations associated with applying complementary high-throughput molecular technologies to address important ecological questions.« less

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

NASA Astrophysics Data System (ADS)

So, Peter T.

2016-03-01

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

Structural Genomics and Drug Discovery for Infectious Diseases

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

Anderson, W.F.

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

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

NASA Astrophysics Data System (ADS)

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

2017-11-01

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

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

PubMed

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

2015-02-01

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

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

Federal Register 2010, 2011, 2012, 2013, 2014

2011-01-28

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

ClusCo: clustering and comparison of protein models.

PubMed

Jamroz, Michal; Kolinski, Andrzej

2013-02-22

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

Characterization of Bacterial Community Dynamics during the Decomposition of Pig Carcasses in Simulated Soil Burial and Composting Systems.

PubMed

Ki, Bo-Min; Kim, Yu Mi; Jeon, Jun Min; Ryu, Hee Wook; Cho, Kyung-Suk

2017-12-28

Soil burial is the most widely used disposal method for infected pig carcasses, but composting has gained attention as an alternative disposal method because pig carcasses can be decomposed rapidly and safely by composting. To understand the pig carcass decomposition process in soil burial and by composting, pilot-scale test systems that simulated soil burial and composting were designed and constructed in the field. The envelope material samples were collected using special sampling devices without disturbance, and bacterial community dynamics were analyzed by high-throughput pyrosequencing for 340 days. Based on the odor gas intensity profiles, it was estimated that the active and advanced decay stages were reached earlier by composting than by soil burial. The dominant bacterial communities in the soil were aerobic and/or facultatively anaerobic gram-negative bacteria such as Pseudomonas, Gelidibacter, Mucilaginibacter , and Brevundimonas . However, the dominant bacteria in the composting system were anaerobic, thermophilic, endospore-forming, and/or halophilic gram-positive bacteria such as Pelotomaculum, Lentibacillus, Clostridium , and Caldicoprobacter . Different dominant bacteria played important roles in the decomposition of pig carcasses in the soil and compost. This study provides useful comparative date for the degradation of pig carcasses in the soil burial and composting systems.

Bacterial Community Diversity of Oil-Contaminated Soils Assessed by High Throughput Sequencing of 16S rRNA Genes.

PubMed

Peng, Mu; Zi, Xiaoxue; Wang, Qiuyu

2015-09-24

Soil bacteria play a major role in ecological and biodegradable function processes in oil-contaminated soils. Here, we assessed the bacterial diversity and changes therein in oil-contaminated soils exposed to different periods of oil pollution using 454 pyrosequencing of 16S rRNA genes. No less than 24,953 valid reads and 6246 operational taxonomic units (OTUs) were obtained from all five studied samples. OTU richness was relatively higher in contaminated soils than clean samples. Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Planctomycetes and Proteobacteria were the dominant phyla among all the soil samples. The heatmap plot depicted the relative percentage of each bacterial family within each sample and clustered five samples into two groups. For the samples, bacteria in the soils varied at different periods of oil exposure. The oil pollution exerted strong selective pressure to propagate many potentially petroleum degrading bacteria. Redundancy analysis (RDA) indicated that organic matter was the highest determinant factor for explaining the variations in community compositions. This suggests that compared to clean soils, oil-polluted soils support more diverse bacterial communities and soil bacterial community shifts were mainly controlled by organic matter and exposure time. These results provide some useful information for bioremediation of petroleum contaminated soil in the future.

Impacts of infection with different toxigenic Clostridium difficile strains on faecal microbiota in children

NASA Astrophysics Data System (ADS)

Ling, Zongxin; Liu, Xia; Jia, Xiaoyun; Cheng, Yiwen; Luo, Yueqiu; Yuan, Li; Wang, Yuezhu; Zhao, Chunna; Guo, Shu; Li, Lanjuan; Xu, Xiwei; Xiang, Charlie

2014-12-01

Increasing evidence suggests that altered intestinal microbial composition and function result in an increased risk of Clostridium difficile-associated diarrhoea (CDAD); however, the specific changes of intestinal microbiota in children suffering from CDAD and their associations with C. difficile strain toxigenicity are poorly understood. High-throughput pyrosequencing showed that reduced faecal bacterial diversity and dramatic shifts of microbial composition were found in children with CDAD. The Firmicutes/Bacteroidetes ratio was increased significantly in patients with CDAD, which indicated that dysbiosis of faecal microbiota was closely associated with CDAD. C. difficile infection resulted in an increase in lactate-producing phylotypes, with a corresponding decrease in butyrate-producing bacteria. The decrease in butyrate and lactate buildup impaired intestinal colonisation resistance, which increased the susceptibility to C. difficile colonisation. Strains of C. difficile which were positive for both toxin A and toxin B reduced faecal bacterial diversity to a greater degree than strains that were only toxin B-positive, and were associated with unusually abundant Enterococcus, which implies that the C. difficile toxins have different impacts on the faecal microbiota of children. Greater understanding of the relationships between disruption of the normal faecal microbiota and colonisation with C. difficile that produces different toxins might lead to improved treatment.

Lentinula edodes-derived polysaccharide rejuvenates mice in terms of immune responses and gut microbiota.

PubMed

Xu, Xiaofei; Yang, Jiguo; Ning, Zhengxiang; Zhang, Xuewu

2015-08-01

Aging is characterized by impaired immunity and unbalanced gut microbiota. Prebiotics have the capability to prevent or reverse age-related declines in health by modulating gut microbiota. Mushroom polysaccharides have been suggested to be potential prebiotics. However, their effects on the immunity and gut microbiota in aged mice have not been determined. This study firstly assessed the effects of a heteropolysaccharide L2 isolated from the fruit body of L. edodes on the immune response of aged mice, and then compared the composition of fecal microbiota in adult (N), old (O) and L2-treated old (Oa) mice using the high-throughput pyrosequencing technique. The results showed that L2 can restore the age-attenuated immune responses by increasing cytokine levels in peripheral blood. Moreover, L2 can partly reverse the age-altered composition of gut microbiota. The Euclidean distances (De) among 3 groups (N, O and Oa) are determined to be De(O, N) = 0.19, De(O, Oa) = 0.20, and De(N, Oa) = 0.10, i.e. there is a marked reduction in the distance from 0.19 to 0.1 by L2. This suggests the beneficial effects of L2 on enhancing immunity and improving gut health.

Soil Bacterial Community Shifts after Chitin Enrichment: An Integrative Metagenomic Approach

PubMed Central

Jacquiod, Samuel; Franqueville, Laure; Cécillon, Sébastien; M. Vogel, Timothy; Simonet, Pascal

2013-01-01

Chitin is the second most produced biopolymer on Earth after cellulose. Chitin degrading enzymes are promising but untapped sources for developing novel industrial biocatalysts. Hidden amongst uncultivated micro-organisms, new bacterial enzymes can be discovered and exploited by metagenomic approaches through extensive cloning and screening. Enrichment is also a well-known strategy, as it allows selection of organisms adapted to feed on a specific compound. In this study, we investigated how the soil bacterial community responded to chitin enrichment in a microcosm experiment. An integrative metagenomic approach coupling phylochips and high throughput shotgun pyrosequencing was established in order to assess the taxonomical and functional changes in the soil bacterial community. Results indicate that chitin enrichment leads to an increase of Actinobacteria, γ-proteobacteria and β-proteobacteria suggesting specific selection of chitin degrading bacteria belonging to these classes. Part of enriched bacterial genera were not yet reported to be involved in chitin degradation, like the members from the Micrococcineae sub-order (Actinobacteria). An increase of the observed bacterial diversity was noticed, with detection of specific genera only in chitin treated conditions. The relative proportion of metagenomic sequences related to chitin degradation was significantly increased, even if it represents only a tiny fraction of the sequence diversity found in a soil metagenome. PMID:24278158

Reestablishment of recipient-associated microbiota in the lung allograft is linked to reduced risk of bronchiolitis obliterans syndrome.

PubMed

Willner, Dana L; Hugenholtz, Philip; Yerkovich, Stephanie T; Tan, Maxine E; Daly, Joshua N; Lachner, Nancy; Hopkins, Peter M; Chambers, Daniel C

2013-03-15

Bronchiolitis obliterans syndrome (BOS) is the primary limiting factor for long-term survival after lung transplantation, and has previously been associated with microbial infections. To cross-sectionally and longitudinally characterize microbial communities in allografts from transplant recipients with and without BOS using a culture-independent method based on high-throughput sequencing. Allografts were sampled by bronchoalveolar lavage, and microbial communities were profiled using 16S rRNA gene amplicon pyrosequencing. Community profiles were compared using the weighted Unifrac metric and the relationship between microbial populations, BOS, and other covariates was explored using PERMANOVA and logistic regression. Microbial communities in transplant patients fell into two main groups: those dominated by Pseudomonas or those dominated by Streptococcus and Veillonella, which seem to be mutually exclusive lung microbiomes. Aspergillus culture was also negatively correlated with the Pseudomonas-dominated group. The reestablishment of dominant populations present in patients pretransplant, notably Pseudomonas in individuals with cystic fibrosis, was negatively correlated with BOS. Recolonization of the allograft by Pseudomonas in individuals with cystic fibrosis is not associated with BOS. In general, reestablishment of pretransplant lung populations in the allograft seems to have a protective effect against BOS, whereas de novo acquisition of microbial populations often belonging to the same genera may increase the risk of BOS.

New approach for the study of mite reproduction: The first transcriptome analysis of a mite, Phytoseiulus persimilis (Acari: Phytoseiidae).

PubMed

Cabrera, Ana R; Donohue, Kevin V; Khalil, Sayed M S; Scholl, Elizabeth; Opperman, Charles; Sonenshine, Daniel E; Roe, R Michael

2011-01-01

Many species of mites and ticks are of agricultural and medical importance. Much can be learned from the study of transcriptomes of acarines which can generate DNA-sequence information of potential target genes for the control of acarine pests. High throughput transcriptome sequencing can also yield sequences of genes critical during physiological processes poorly understood in acarines, i.e., the regulation of female reproduction in mites. The predatory mite, Phytoseiulus persimilis, was selected to conduct a transcriptome analysis using 454 pyrosequencing. The objective of this project was to obtain DNA-sequence information of expressed genes from P. persimilis with special interest in sequences corresponding to vitellogenin (Vg) and the vitellogenin receptor (VgR). These genes are critical to the understanding of vitellogenesis, and they will facilitate the study of the regulation of mite female reproduction. A total of 12,556 contiguous sequences (contigs) were assembled with an average size of 935bp. From these sequences, the putative translated peptides of 11 contigs were similar in amino acid sequences to other arthropod Vgs, while 6 were similar to VgRs. We selected some of these sequences to conduct stage-specific expression studies to further determine their function. 2010 Elsevier Ltd. All rights reserved.

The Rebirth of Culture in Microbiology through the Example of Culturomics To Study Human Gut Microbiota

PubMed Central

Lagier, Jean-Christophe; Hugon, Perrine; Khelaifia, Saber; Fournier, Pierre-Edouard; La Scola, Bernard

2015-01-01

SUMMARY Bacterial culture was the first method used to describe the human microbiota, but this method is considered outdated by many researchers. Metagenomics studies have since been applied to clinical microbiology; however, a “dark matter” of prokaryotes, which corresponds to a hole in our knowledge and includes minority bacterial populations, is not elucidated by these studies. By replicating the natural environment, environmental microbiologists were the first to reduce the “great plate count anomaly,” which corresponds to the difference between microscopic and culture counts. The revolution in bacterial identification also allowed rapid progress. 16S rRNA bacterial identification allowed the accurate identification of new species. Mass spectrometry allowed the high-throughput identification of rare species and the detection of new species. By using these methods and by increasing the number of culture conditions, culturomics allowed the extension of the known human gut repertoire to levels equivalent to those of pyrosequencing. Finally, taxonogenomics strategies became an emerging method for describing new species, associating the genome sequence of the bacteria systematically. We provide a comprehensive review on these topics, demonstrating that both empirical and hypothesis-driven approaches will enable a rapid increase in the identification of the human prokaryote repertoire. PMID:25567229

Transcriptome-wide investigation of genomic imprinting in chicken

PubMed Central

Frésard, Laure; Leroux, Sophie; Servin, Bertrand; Gourichon, David; Dehais, Patrice; Cristobal, Magali San; Marsaud, Nathalie; Vignoles, Florence; Bed'hom, Bertrand; Coville, Jean-Luc; Hormozdiari, Farhad; Beaumont, Catherine; Zerjal, Tatiana; Vignal, Alain; Morisson, Mireille; Lagarrigue, Sandrine; Pitel, Frédérique

2014-01-01

Genomic imprinting is an epigenetic mechanism by which alleles of some specific genes are expressed in a parent-of-origin manner. It has been observed in mammals and marsupials, but not in birds. Until now, only a few genes orthologous to mammalian imprinted ones have been analyzed in chicken and did not demonstrate any evidence of imprinting in this species. However, several published observations such as imprinted-like QTL in poultry or reciprocal effects keep the question open. Our main objective was thus to screen the entire chicken genome for parental-allele-specific differential expression on whole embryonic transcriptomes, using high-throughput sequencing. To identify the parental origin of each observed haplotype, two chicken experimental populations were used, as inbred and as genetically distant as possible. Two families were produced from two reciprocal crosses. Transcripts from 20 embryos were sequenced using NGS technology, producing ∼200 Gb of sequences. This allowed the detection of 79 potentially imprinted SNPs, through an analysis method that we validated by detecting imprinting from mouse data already published. However, out of 23 candidates tested by pyrosequencing, none could be confirmed. These results come together, without a priori, with previous statements and phylogenetic considerations assessing the absence of genomic imprinting in chicken. PMID:24452801

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

PubMed

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

2010-07-01

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

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

PubMed Central

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

2010-01-01

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

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

PubMed

Alexander, Nathan; Woetzel, Nils; Meiler, Jens

2011-02-01

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

Validation of the VE1 Immunostain for the BRAF V600E Mutation in Melanoma

PubMed Central

Pearlstein, Michelle V.; Zedek, Daniel C.; Ollila, David W.; Treece, Amanda; Gulley, Margaret L.; Groben, Pamela A.; Thomas, Nancy E.

2014-01-01

BACKGROUND BRAF mutation status, and therefore eligibility for BRAF inhibitors, is currently determined by sequencing methods. We assessed the validity of VE1, a monoclonal antibody against the BRAF V600E mutant protein, in the detection of mutant BRAF V600E melanomas as classified by DNA pyrosequencing. METHODS The cases were 76 metastatic melanoma patients with only one known primary melanoma who had had BRAF codon 600 pyrosequencing of either their primary (n=19), metastatic (n=57) melanoma, or both (n=17). All melanomas (n=93) were immunostained with the BRAF VE1 antibody using a red detection system. The staining intensity of these specimens was scored from 0 – 3+ by a dermatopathologist. Scores of 0 and 1+ were considered as negative staining while scores of 2+ and 3+ were considered positive. RESULTS The VE1 antibody demonstrated a sensitivity of 85% and a specificity of 100% as compared to DNA pyrosequencing results. There was 100% concordance between VE1 immunostaining of primary and metastatic melanomas from the same patient. V600K, V600Q, and V600R BRAF melanomas did not positively stain with VE1. CONCLUSIONS This hospital-based study finds high sensitivity and specificity for the BRAF VE1 immunostain in comparison to pyrosequencing in detection of BRAF V600E in melanomas. PMID:24917033

Massively parallel pyrosequencing-based transcriptome analyses of small brown planthopper (Laodelphax striatellus), a vector insect transmitting rice stripe virus (RSV)

PubMed Central

2010-01-01

Background The small brown planthopper (Laodelphax striatellus) is an important agricultural pest that not only damages rice plants by sap-sucking, but also acts as a vector that transmits rice stripe virus (RSV), which can cause even more serious yield loss. Despite being a model organism for studying entomology, population biology, plant protection, molecular interactions among plants, viruses and insects, only a few genomic sequences are available for this species. To investigate its transcriptome and determine the differences between viruliferous and naïve L. striatellus, we employed 454-FLX high-throughput pyrosequencing to generate EST databases of this insect. Results We obtained 201,281 and 218,681 high-quality reads from viruliferous and naïve L. striatellus, respectively, with an average read length as 230 bp. These reads were assembled into contigs and two EST databases were generated. When all reads were combined, 16,885 contigs and 24,607 singletons (a total of 41,492 unigenes) were obtained, which represents a transcriptome of the insect. BlastX search against the NCBI-NR database revealed that only 6,873 (16.6%) of these unigenes have significant matches. Comparison of the distribution of GO classification among viruliferous, naïve, and combined EST databases indicated that these libraries are broadly representative of the L. striatellus transcriptomes. Functionally diverse transcripts from RSV, endosymbiotic bacteria Wolbachia and yeast-like symbiotes were identified, which reflects the possible lifestyles of these microbial symbionts that live in the cells of the host insect. Comparative genomic analysis revealed that L. striatellus encodes similar innate immunity regulatory systems as other insects, such as RNA interference, JAK/STAT and partial Imd cascades, which might be involved in defense against viral infection. In addition, we determined the differences in gene expression between vector and naïve samples, which generated a list of candidate genes that are potentially involved in the symbiosis of L. striatellus and RSV. Conclusions To our knowledge, the present study is the first description of a genomic project for L. striatellus. The identification of transcripts from RSV, Wolbachia, yeast-like symbiotes and genes abundantly expressed in viruliferous insect, provided a starting-point for investigating the molecular basis of symbiosis among these organisms. PMID:20462456

A pyrosequencing assay for the quantitative methylation analysis of the PCDHB gene cluster, the major factor in neuroblastoma methylator phenotype.

PubMed

Banelli, Barbara; Brigati, Claudio; Di Vinci, Angela; Casciano, Ida; Forlani, Alessandra; Borzì, Luana; Allemanni, Giorgio; Romani, Massimo

2012-03-01

Epigenetic alterations are hallmarks of cancer and powerful biomarkers, whose clinical utilization is made difficult by the absence of standardization and of common methods of data interpretation. The coordinate methylation of many loci in cancer is defined as 'CpG island methylator phenotype' (CIMP) and identifies clinically distinct groups of patients. In neuroblastoma (NB), CIMP is defined by a methylation signature, which includes different loci, but its predictive power on outcome is entirely recapitulated by the PCDHB cluster only. We have developed a robust and cost-effective pyrosequencing-based assay that could facilitate the clinical application of CIMP in NB. This assay permits the unbiased simultaneous amplification and sequencing of 17 out of 19 genes of the PCDHB cluster for quantitative methylation analysis, taking into account all the sequence variations. As some of these variations were at CpG doublets, we bypassed the data interpretation conducted by the methylation analysis software to assign the corrected methylation value at these sites. The final result of the assay is the mean methylation level of 17 gene fragments in the protocadherin B cluster (PCDHB) cluster. We have utilized this assay to compare the methylation levels of the PCDHB cluster between high-risk and very low-risk NB patients, confirming the predictive value of CIMP. Our results demonstrate that the pyrosequencing-based assay herein described is a powerful instrument for the analysis of this gene cluster that may simplify the data comparison between different laboratories and, in perspective, could facilitate its clinical application. Furthermore, our results demonstrate that, in principle, pyrosequencing can be efficiently utilized for the methylation analysis of gene clusters with high internal homologies.

Somatic profiling of the epidermal growth factor receptor pathway in tumours from patients with advanced colorectal cancer, treated with chemotherapy ± cetuximab

PubMed Central

Smith, Christopher G.; Fisher, David; Claes, Bart; Maughan, Timothy S.; Idziaszczyk, Shelley; Peuteman, Gilian; Harris, Rebecca; James, Michelle D.; Meade, Angela; Jasani, Bharat; Adams, Richard A.; Kenny, Sarah; Kaplan, Richard; Lambrechts, Diether; Cheadle, Jeremy P.

2013-01-01

Purpose To study the somatic molecular profile of the epidermal growth factor receptor (EGFR) pathway in advanced CRC (aCRC), its relationship to prognosis, the site of the primary and metastases, and response to cetuximab. Experimental Design We used Sequenom and Pyrosequencing for high-throughput somatic profiling the EGFR pathway in 1,976 tumours from patients with aCRC from the COIN trial (oxaliplatin and fluoropyrimidine chemotherapy ±cetuximab). Correlations between mutations, clinico-pathological, response and survival data were carried out. Results Sequenom and Pyrosequencing had 99.0% (9961/10063) genotype concordance. We identified thirteen different KRAS mutations in 42.3% of aCRCs, two BRAF mutations in 9.0%, four NRAS mutations in 3.6% and five PIK3CA mutations in 12.7%. 4.2% of aCRCs had microsatellite instability (MSI). KRAS and PIK3CA exon 9, but not exon 20, mutations co-occurred (P=8.9×10−4) as did MSI and BRAF mutations (P=5.3×10−10). KRAS mutations were associated with right colon cancers (P=5.2×10−5) and BRAF mutations with right (P=7.2×10−5) and transverse colon (P=9.8×10−6) cancers. KRAS mutations were associated with lung-only metastases (P=2.3×10−4), BRAF mutations with peritoneal (P=9.2×10−4) and nodal-only (P=3.7×10−5) metastases, and MSI (BRAFWT) with nodal-only metastases (P=2.9×10−4). MSI (BRAFWT) was associated with worse survival (HR=1.89, 95% CI 1.30-2.76, P=8.5×10−4). No mutations, subsets of mutations, or MSI-status were associated with response to cetuximab. Conclusions Our data support a functional co-operation between KRAS and PIK3CA in colorectal tumourigenesis and link somatic profiles to the sites of metastases. MSI was associated with poor prognosis in advanced disease, and no individual somatic profile was associated with response to cetuximab in COIN. PMID:23741067

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

PubMed Central

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

2011-01-01

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 can effectively breakdown a wide variety of complex polysaccharides. PMID:21451584

Experimental induction of proventricular dilatation disease in cockatiels (Nymphicus hollandicus) inoculated with brain homogenates containing avian bornavirus 4

PubMed Central

Gancz, Ady Y; Kistler, Amy L; Greninger, Alexander L; Farnoushi, Yigal; Mechani, Sara; Perl, Shmuel; Berkowitz, Asaf; Perez, Noa; Clubb, Susan; DeRisi, Joseph L; Ganem, Don; Lublin, Avishai

2009-01-01

Background Proventricular dilatation disease (PDD) is a fatal disorder of psittacine birds worldwide. The disease is characterized by lymphoplasmacytic infiltration of the central and peripheral nervous systems, leading to gastrointestinal motility and/or central nervous system dysfunction. Recently, we detected a significant association between avian bornavirus (ABV) infection and clinical signs of PDD in psittacines. However, it remains unclear whether ABV infection actually causes PDD. To address this question, we examined the impact of ABV inoculation on the cockatiel (Nymphicus hollandicus). Results Five cockatiels were inoculated via multiple routes (intramuscular, intraocular, intranasal, and oral) with a brain homogenate derived from either a PDD(+) avian bornavirus 4 (ABV4) (+) case (n = 3 inoculees) or from a PDD(-) ABV(-) control (n = 2 inoculees). The control birds remained free of clinical or pathological signs of PDD, and tested ABV(-) by RT-PCR and immunohistochemistry (IHC). In contrast, all three cockatiels inoculated with ABV4(+) brain homogenate developed gross and microscopic PDD lesions, and two exhibited overt clinical signs. In numerous tissues, ABV RT-PCR and sequence analysis demonstrated the presence of ABV4 RNA nearly identical to that in the inoculum. ABV was detected in the central nervous system of the three ABV-inoculees by IHC. Pyrosequencing to investigate the viral flora in the ABV4(+) inoculum uncovered 7 unique reads sharing 73–100% nucleotide sequence identity with previously identified ABV sequences and 24 reads sharing 40–89% amino acid sequence identity with viruses in the Retroviridae and Astroviridae families. Of these candidate viral species, only ABV RNA was recovered from tissues of the inoculated birds. Conclusion In this study, the clinical and pathological manifestations of PDD were induced by inoculation of cockatiels with brain homogenates containing avian bornavirus 4. By using high throughput pyrosequencing an in-depth view of the viral content of the inoculum was achieved, revealing that of 3 candidate virus families detected, only the presence of ABV RNA correlated with the development of PDD. This study provides evidence of a causal association between ABV4 infection and PDD in cockatiels. PMID:19589169

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

USDA-ARS?s Scientific Manuscript database

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

Evaluation of six primer pairs targeting the nuclear rRNA operon for characterization of arbuscular mycorrhizal fungal (AMF) communities using 454 pyrosequencing.

PubMed

Van Geel, Maarten; Busschaert, Pieter; Honnay, Olivier; Lievens, Bart

2014-11-01

In the last few years, 454 pyrosequencing-based analysis of arbuscular mycorrhizal fungal (AMF; Glomeromycota) communities has tremendously increased our knowledge of the distribution and diversity of AMF. Nonetheless, comparing results between different studies is difficult, as different target genes (or regions thereof) and primer combinations, with potentially dissimilar specificities and efficacies, are being utilized. In this study we evaluated six primer pairs that have previously been used in AMF studies (NS31-AM1, AMV4.5NF-AMDGR, AML1-AML2, NS31-AML2, FLR3-LSUmBr and Glo454-NDL22) for their use in 454 pyrosequencing based on both an in silico approach and 454 pyrosequencing of AMF communities from apple tree roots. Primers were evaluated in terms of (i) in silico coverage of Glomeromycota fungi, (ii) the number of high-quality sequences obtained, (iii) selectivity for AMF species, (iv) reproducibility and (v) ability to accurately describe AMF communities. We show that primer pairs AMV4.5NF-AMDGR, AML1-AML2 and NS31-AML2 outperformed the other tested primer pairs in terms of number of Glomeromycota reads (AMF specificity and coverage). Additionally, these primer pairs were found to have no or only few mismatches to AMF sequences and were able to consistently describe AMF communities from apple roots. However, whereas most high-quality AMF sequences were obtained for AMV4.5NF-AMDGR, our results also suggest that this primer pair favored amplification of Glomeraceae sequences at the expense of Ambisporaceae, Claroideoglomeraceae and Paraglomeraceae sequences. Furthermore, we demonstrate the complementary specificity of AMV4.5NF-AMDGR with AML1-AML2, and of AMV4.5NF-AMDGR with NS31-AML2, making these primer combinations highly suitable for tandem use in covering the diversity of AMF communities. Copyright © 2014 Elsevier B.V. All rights reserved.

Pyrosequencing the Bemisia tabaci Transcriptome Reveals a Highly Diverse Bacterial Community and a Robust System for Insecticide Resistance

PubMed Central

Wu, Qing-jun; Wang, Shao-li; Yang, Xin; Yang, Ni-na; Li, Ru-mei; Jiao, Xiao-guo; Pan, Hui-peng; Liu, Bai-ming; Su, Qi; Xu, Bao-yun; Hu, Song-nian; Zhou, Xu-guo; Zhang, You-jun

2012-01-01

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 B. tabaci complex. Moreover, current pyrosequencing effort greatly enriched the existing whitefly EST database, and makes RNAseq a viable option for future genomic analysis. PMID:22558125

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

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

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

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

Combinatorial and High Throughput Discovery of High Temperature Piezoelectric Ceramics

DTIC Science & Technology

2011-10-10

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

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

EPA Science Inventory

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

Focused Evolution of HIV-1 Neutralizing Antibodies Revealed by Structures and Deep Sequencing

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

Wu, Xueling; Zhou, Tongqing; Zhu, Jiang

2013-03-04

Antibody VRC01 is a human immunoglobulin that neutralizes about 90% of HIV-1 isolates. To understand how such broadly neutralizing antibodies develop, we used x-ray crystallography and 454 pyrosequencing to characterize additional VRC01-like antibodies from HIV-1-infected individuals. Crystal structures revealed a convergent mode of binding for diverse antibodies to the same CD4-binding-site epitope. A functional genomics analysis of expressed heavy and light chains revealed common pathways of antibody-heavy chain maturation, confined to the IGHV1-2*02 lineage, involving dozens of somatic changes, and capable of pairing with different light chains. Broadly neutralizing HIV-1 immunity associated with VRC01-like antibodies thus involves the evolution ofmore » antibodies to a highly affinity-matured state required to recognize an invariant viral structure, with lineages defined from thousands of sequences providing a genetic roadmap of their development.« less

Pyrosequencing reveals the key microorganisms involved in sludge alkaline fermentation for efficient short-chain fatty acids production.

PubMed

Zheng, Xiong; Su, Yinglong; Li, Xiang; Xiao, Naidong; Wang, Dongbo; Chen, Yinguang

2013-05-07

Short-chain fatty acids (SCFAs) have been regarded as the excellent carbon source of wastewater biological nutrient removal, and sludge alkaline (pH 10) fermentation has been reported to achieve highly efficient SCFAs production. In this study, the underlying mechanisms for the improved SCFAs production at pH 10 were investigated by using 454 pyrosequencing and fluorescent in situ hybridization (FISH) to analyze the microbial community structures in sludge fermentation reactors. It was found that sludge fermentation at pH 10 increased the abundances of Pseudomonas sp. and Alcaligenes sp., which were able to excrete extracellular proteases and depolymerases, and thus enhanced the hydrolysis of insoluble sludge protein and polyhydroxyalkanoates (PHA). Meanwhile, the abundance of acid-producing bacteria (such as Clostridium sp.) in the reactor of pH 10 was also higher than that of uncontrolled pH, which benefited the acidification of soluble organic substrates. Further study indicated that sludge fermentation at pH 10 significantly decreased the number of methanogenic archaea, resulting in lower SCFAs consumption and lower methane production. Therefore, anaerobic sludge fermentation under alkaline conditions increased the abundances of bacteria involved in sludge hydrolysis and acidification, and decreased the abundance of methanogenic archaea, which favored the competition of bacteria over methanogens and resulted in the efficient production of SCFAs.

Molecular Analysis of Bacterial Communities in Biofilms of a Drinking Water Clearwell

PubMed Central

Zhang, Minglu; Liu, Wenjun; Nie, Xuebiao; Li, Cuiping; Gu, Junnong; Zhang, Can

2012-01-01

Microbial community structures in biofilms of a clearwell in a drinking water supply system in Beijing, China were examined by clone library, terminal restriction fragment length polymorphism (T-RFLP) and 454 pyrosequencing of the amplified 16S rRNA gene. Six biofilm samples (designated R1–R6) collected from six locations (upper and lower sites of the inlet, middle and outlet) of the clearwell revealed similar bacterial patterns by T-RFLP analysis. With respect to the dominant groups, the phylotypes detected by clone library and T-RFLP generally matched each other. A total of 9,543 reads were obtained from samples located at the lower inlet and the lower outlet sites by pyrosequencing. The bacterial diversity of the two samples was compared at phylum and genus levels. Alphaproteobacteria dominated the communities in both samples and the genus of Sphingomonas constituted 75.1%–99.6% of this phylum. A high level of Sphingomonas sp. was first observed in the drinking water biofilms with 0.6–1.0 mg L−1 of chlorine residual. Disinfectant-resistant microorganisms deserve special attention in drinking water management. This study provides novel insights into the microbial populations in drinking water systems and highlights the important role of Sphingomonas species in biofilm formation. PMID:23059725

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

PubMed

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.

Denaturing Gradient Gel Electrophoresis and Barcoded Pyrosequencing Reveal Unprecedented Archaeal Diversity in Mangrove Sediment and Rhizosphere Samples

PubMed Central

Pires, Ana C. C.; Cleary, Daniel F. R.; Almeida, Adelaide; Cunha, Ângela; Dealtry, Simone; Mendonça-Hagler, Leda C. S.; Smalla, Kornelia

2012-01-01

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. PMID:22660713

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

NASA Astrophysics Data System (ADS)

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

2016-12-01

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

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

PubMed

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

2017-10-01

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

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

PubMed

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

2013-05-01

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

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

PubMed Central

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

2014-01-01

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

High-Throughput, Data-Rich Cellular RNA Device Engineering

PubMed Central

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

2015-01-01

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

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

PubMed Central

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

2014-01-01

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

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

EPA Science Inventory

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

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

Ottesen, Elizabeth A.; Marin, Roman; Preston, Christina M.

Planktonic microbial activity and community structure is dynamic, and can change dramatically on time scales of hours to days. Yet for logistical reasons, this temporal scale is typically undersampled in the marine environment. In order to facilitate higher-resolution, long-term observation of microbial diversity and activity, we developed a protocol for automated collection and fixation of marine microbes using the Environmental Sample Processor (ESP) platform. The protocol applies a preservative (RNALater) to cells collected on filters, for long-term storage and preservation of total cellular RNA. Microbial samples preserved using this protocol yielded high-quality RNA after 30 days of storage at roommore » temperature, or onboard the ESP at in situ temperatures. Pyrosequencing of complementary DNA libraries generated from ESP-collected and preserved samples yielded transcript abundance profiles nearly indistinguishable from those derived from conventionally treated replicate samples. To demonstrate the utility of the method, we used a moored ESP to remotely and autonomously collect Monterey Bay seawater for metatranscriptomic analysis. Community RNA was extracted and pyrosequenced from samples collected at four time points over the course of a single day. In all four samples, the oxygenic photoautotrophs were predominantly eukaryotic, while the bacterial community was dominated by Polaribacter-like Flavobacteria and a Rhodobacterales bacterium sharing high similarity with Rhodobacterales sp. HTCC2255. However, each time point was associated with distinct species abundance and gene transcript profiles. These laboratory and field tests confirmed that autonomous collection and preservation is a feasible and useful approach for characterizing the expressed genes and environmental responses of marine microbial communities.« less

Molecular techniques revealed highly diverse microbial communities in natural marine biofilms on polystyrene dishes for invertebrate larval settlement.

PubMed

Lee, On On; Chung, Hong Chun; Yang, Jiangke; Wang, Yong; Dash, Swagatika; Wang, Hao; Qian, Pei-Yuan

2014-07-01

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.

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

NASA Astrophysics Data System (ADS)

Hayasaki, Yoshio

2017-02-01

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

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

PubMed

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

2017-06-20

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

Time-dependent effect of graphene on the structure, abundance, and function of the soil bacterial community.

PubMed

Ren, Wenjie; Ren, Gaidi; Teng, Ying; Li, Zhengao; Li, Lina

2015-10-30

The increased application of graphene raises concerns about its environmental impact, but little information is available on the effect of graphene on the soil microbial community. This study evaluated the impact of graphene on the structure, abundance and function of the soil bacterial community based on quantitative real-time polymerase chain reaction (qPCR), pyrosequencing and soil enzyme activities. The results show that the enzyme activities of dehydrogenase and fluorescein diacetate (FDA) esterase and the biomass of the bacterial populations were transiently promoted by the presence of graphene after 4 days of exposure, but these parameters recovered completely after 21 days. Pyrosequencing analysis suggested a significant shift in some bacterial populations after 4 days, and the shift became weaker or disappeared as the exposure time increased to 60 days. During the entire exposure process, the majority of bacterial phylotypes remained unaffected. Some bacterial populations involved in nitrogen biogeochemical cycles and the degradation of organic compounds can be affected by the presence of graphene. Copyright © 2015 Elsevier B.V. All rights reserved.

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

PubMed

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

2015-01-01

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

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

EPA Science Inventory

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

Melatonin prevents obesity through modulation of gut microbiota in mice.

PubMed

Xu, Pengfei; Wang, Jialin; Hong, Fan; Wang, Sheng; Jin, Xi; Xue, Tingting; Jia, Li; Zhai, Yonggong

2017-05-01

Excess weight and obesity are severe public health threats worldwide. Recent evidence demonstrates that gut microbiota dysbiosis contributes to obesity and its comorbidities. The body weight-reducing and energy balancing effects of melatonin have been reported in several studies, but to date, no investigations toward examining whether the beneficial effects of melatonin are associated with gut microbiota have been carried out. In this study, we show that melatonin reduces body weight, liver steatosis, and low-grade inflammation as well as improving insulin resistance in high fat diet (HFD)-fed mice. High-throughput pyrosequencing of the 16S rRNA demonstrated that melatonin treatment significantly changed the composition of the gut microbiota in mice fed an HFD. The richness and diversity of gut microbiota were notably decreased by melatonin. HFD feeding altered 69 operational taxonomic units (OTUs) compare with a normal chow diet (NCD) group, and melatonin supplementation reversed 14 OTUs to the same configuration than those present in the NCD group, thereby impacting various functions, in particular through its ability to decrease the Firmicutes-to-Bacteroidetes ratio and increase the abundance of mucin-degrading bacteria Akkermansia, which is associated with healthy mucosa. Taken together, our results suggest that melatonin may be used as a probiotic agent to reverse HFD-induced gut microbiota dysbiosis and help us to gain a better understanding of the mechanisms governing the various melatonin beneficial effects. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Bacterial communities and their association with the bio-drying of sewage sludge.

PubMed

Cai, Lu; Chen, Tong-Bin; Gao, Ding; Yu, Jie

2016-03-01

Bio-drying is a technology that aims to remove water from a material using the microbial heat originating from organic matter degradation. However, the evolution of bacterial communities that are associated with the drying process has not been researched systematically. This study was performed to investigate the variations of bacterial communities and the relationships among bacterial communities, water evaporation, water generation, and organic matter degradation during the bio-drying of sewage sludge. High-throughput pyrosequencing was used to analyze the bacterial communities, while water evaporation and water generation were determined based on an in situ water vapor monitoring device. The values of water evaporation, water generation, and volatile solids degradation were 412.9 g kg(-1) sewage sludge bio-drying material (SSBM), 65.0 g kg(-1) SSBM, and 70.2 g kg(-1) SSBM, respectively. Rarefaction curves and diversity indices showed that bacterial diversity plummeted after the temperature of the bio-drying pile dramatically increased on d 2, which coincided with a remarkable increase of water evaporation on d 2. Bacterial diversity increased when the pile cooled. During the thermophilic phase, in which Acinetobacter and Bacillus were the dominant genera, the rates of water evaporation, water generation, and VS degradation peaked. These results implied that the elevated temperature reshaped the bacterial communities, which played a key role in water evaporation, and the high temperature also contributed to the effective elimination of pathogens. Copyright © 2015 Elsevier Ltd. All rights reserved.

The novel polysaccharide deacetylase homologue Pdi contributes to virulence of the aquatic pathogen Streptococcus iniae

PubMed Central

Milani, Carlo J. E.; Aziz, Ramy K.; Locke, Jeffrey B.; Dahesh, Samira; Nizet, Victor; Buchanan, John T.

2010-01-01

The aquatic zoonotic pathogen Streptococcus iniae represents a threat to the worldwide aquaculture industry and poses a risk to humans who handle raw fish. Because little is known about the mechanisms of S. iniae pathogenesis or virulence factors, we established a high-throughput system combining whole-genome pyrosequencing and transposon mutagenesis that allowed us to identify virulence proteins, including Pdi, the polysaccharide deacetylase of S. iniae, that we describe here. Using bioinformatics tools, we identified a highly conserved signature motif in Pdi that is also conserved in the peptidoglycan deacetylase PgdA protein family. A Δpdi mutant was attenuated for virulence in the hybrid striped bass model and for survival in whole fish blood. Moreover, Pdi was found to promote bacterial resistance to lysozyme killing and the ability to adhere to and invade epithelial cells. On the other hand, there was no difference in the autolytic potential, resistance to oxidative killing or resistance to cationic antimicrobial peptides between S. iniae wild-type and Δpdi. In conclusion, we have demonstrated that pdi is involved in S. iniae adherence and invasion, lysozyme resistance and survival in fish blood, and have shown that pdi plays a role in the pathogenesis of S. iniae. Identification of Pdi and other S. iniae virulence proteins is a necessary initial step towards the development of appropriate preventive and therapeutic measures against diseases and economic losses caused by this pathogen. PMID:19762441

Functional metagenomics to mine the human gut microbiome for dietary fiber catabolic enzymes.

PubMed

Tasse, Lena; Bercovici, Juliette; Pizzut-Serin, Sandra; Robe, Patrick; Tap, Julien; Klopp, Christophe; Cantarel, Brandi L; Coutinho, Pedro M; Henrissat, Bernard; Leclerc, Marion; Doré, Joël; Monsan, Pierre; Remaud-Simeon, Magali; Potocki-Veronese, Gabrielle

2010-11-01

The human gut microbiome is a complex ecosystem composed mainly of uncultured bacteria. It plays an essential role in the catabolism of dietary fibers, the part of plant material in our diet that is not metabolized in the upper digestive tract, because the human genome does not encode adequate carbohydrate active enzymes (CAZymes). We describe a multi-step functionally based approach to guide the in-depth pyrosequencing of specific regions of the human gut metagenome encoding the CAZymes involved in dietary fiber breakdown. High-throughput functional screens were first applied to a library covering 5.4 × 10(9) bp of metagenomic DNA, allowing the isolation of 310 clones showing beta-glucanase, hemicellulase, galactanase, amylase, or pectinase activities. Based on the results of refined secondary screens, sequencing efforts were reduced to 0.84 Mb of nonredundant metagenomic DNA, corresponding to 26 clones that were particularly efficient for the degradation of raw plant polysaccharides. Seventy-three CAZymes from 35 different families were discovered. This corresponds to a fivefold target-gene enrichment compared to random sequencing of the human gut metagenome. Thirty-three of these CAZy encoding genes are highly homologous to prevalent genes found in the gut microbiome of at least 20 individuals for whose metagenomic data are available. Moreover, 18 multigenic clusters encoding complementary enzyme activities for plant cell wall degradation were also identified. Gene taxonomic assignment is consistent with horizontal gene transfer events in dominant gut species and provides new insights into the human gut functional trophic chain.

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

DTIC Science & Technology

1983-05-06

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

Pyrosequencing analysis for detection of a BRAFV600E mutation in an FNAB specimen of thyroid nodules.

PubMed

Kim, Suk Kyeong; Kim, Dong-Lim; Han, Hye Seung; Kim, Wan Seop; Kim, Seung Ja; Moon, Won Jin; Oh, Seo Young; Hwang, Tae Sook

2008-06-01

Fine-needle aspiration biopsy (FNAB) is the primary means of distinguishing benign from malignant and of guiding therapeutic intervention in thyroid nodules. However, 10% to 30% of cases with indeterminate cytology in FNAB need other diagnostic tools to refine diagnosis. We compared the pyrosequencing method with the conventional direct DNA sequencing analysis and investigated the usefulness of preoperative BRAF mutation analysis as an adjunct diagnostic tool with routine FNAB. A total of 103 surgically confirmed patients' FNA slides were recruited and DNA was extracted after atypical cells were scraped from the slides. BRAF mutation was analyzed by pyrosequencing and direct DNA sequencing. Sixty-three (77.8%) of 81 histopathologically diagnosed malignant nodules revealed positive BRAF mutation on pyrosequencing analysis. In detail, 63 (84.0%) of 75 papillary thyroid carcinoma (PTC) samples showed positive BRAF mutation, whereas 3 follicular thyroid carcinomas, 1 anaplastic carcinoma, 1 medullary thyroid carcinoma, and 1 metastatic lung carcinoma did not show BRAF mutation. None of 22 benign nodules had BRAF mutation in both pyrosequencing and direct DNA sequencing. Out of 27 thyroid nodules classified as 'indeterminate' on cytologic examination preoperatively, 21 (77.8%) cases turned out to be malignant: 18 PTCs (including 2 follicular variant types) and 3 follicular thyroid carcinomas. Among these, 13 (61.9%) classic PTCs had BRAF mutation. None of 6 benign nodules, including 3 follicular adenomas and 3 nodular hyperplasias, had BRAF mutation. Among 63 PTCs with positive BRAF mutation detected by pyrosequencing analysis, 3 cases did not show BRAF mutation by direct DNA sequencing. Although it was not statistically significant, pyrosequencing was superior to direct DNA sequencing in detecting the BRAF mutation of thyroid nodules (P=0.25). Detecting BRAF mutation by pyrosequencing is more sensitive, faster, and less expensive than direct DNA sequencing and is proposed as an adjunct diagnostic tool in evaluating thyroid nodules of indeterminate cytology.

Methylation profiling identified novel differentially methylated markers including OPCML and FLRT2 in prostate cancer.

PubMed

Wu, Yu; Davison, Jerry; Qu, Xiaoyu; Morrissey, Colm; Storer, Barry; Brown, Lisha; Vessella, Robert; Nelson, Peter; Fang, Min

2016-04-02

To develop new methods to distinguish indolent from aggressive prostate cancers (PCa), we utilized comprehensive high-throughput array-based relative methylation (CHARM) assay to identify differentially methylated regions (DMRs) throughout the genome, including both CpG island (CGI) and non-CGI regions in PCa patients based on Gleason grade. Initially, 26 samples, including 8 each of low [Gleason score (GS) 6] and high (GS ≥7) grade PCa samples and 10 matched normal prostate tissues, were analyzed as a discovery cohort. We identified 3,567 DMRs between normal and cancer tissues, and 913 DMRs distinguishing low from high-grade cancers. Most of these DMRs were located at CGI shores. The top 5 candidate DMRs from the low vs. high Gleason comparison, including OPCML, ELAVL2, EXT1, IRX5, and FLRT2, were validated by pyrosequencing using the discovery cohort. OPCML and FLRT2 were further validated in an independent cohort consisting of 20 low-Gleason and 33 high-Gleason tissues. We then compared patients with biochemical recurrence (n=70) vs. those without (n=86) in a third cohort, and they showed no difference in methylation at these DMR loci. When GS 3+4 cases and GS 4+3 cases were compared, OPCML-DMR methylation showed a trend of lower methylation in the recurrence group (n=30) than in the no-recurrence (n=52) group. We conclude that whole-genome methylation profiling with CHARM revealed distinct patterns of differential DNA methylation between normal prostate and PCa tissues, as well as between different risk groups of PCa as defined by Gleason scores. A panel of selected DMRs may serve as novel surrogate biomarkers for Gleason score in PCa.

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

PubMed Central

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

2007-01-01

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

High mass throughput particle generation using multiple nozzle spraying

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

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

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

High mass throughput particle generation using multiple nozzle spraying

DOEpatents

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

2004-07-20

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

High mass throughput particle generation using multiple nozzle spraying

DOEpatents

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

2009-03-03

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

Fungal Community Structure in Disease Suppressive Soils Assessed by 28S LSU Gene Sequencing

PubMed Central

Penton, C. Ryan; Gupta, V. V. S. R.; Tiedje, James M.; Neate, Stephen M.; Ophel-Keller, Kathy; Gillings, Michael; Harvey, Paul; Pham, Amanda; Roget, David K.

2014-01-01

Natural biological suppression of soil-borne diseases is a function of the activity and composition of soil microbial communities. Soil microbe and phytopathogen interactions can occur prior to crop sowing and/or in the rhizosphere, subsequently influencing both plant growth and productivity. Research on suppressive microbial communities has concentrated on bacteria although fungi can also influence soil-borne disease. Fungi were analyzed in co-located soils ‘suppressive’ or ‘non-suppressive’ for disease caused by Rhizoctonia solani AG 8 at two sites in South Australia using 454 pyrosequencing targeting the fungal 28S LSU rRNA gene. DNA was extracted from a minimum of 125 g of soil per replicate to reduce the micro-scale community variability, and from soil samples taken at sowing and from the rhizosphere at 7 weeks to cover the peak Rhizoctonia infection period. A total of ∼994,000 reads were classified into 917 genera covering 54% of the RDP Fungal Classifier database, a high diversity for an alkaline, low organic matter soil. Statistical analyses and community ordinations revealed significant differences in fungal community composition between suppressive and non-suppressive soil and between soil type/location. The majority of differences associated with suppressive soils were attributed to less than 40 genera including a number of endophytic species with plant pathogen suppression potentials and mycoparasites such as Xylaria spp. Non-suppressive soils were dominated by Alternaria, Gibberella and Penicillum. Pyrosequencing generated a detailed description of fungal community structure and identified candidate taxa that may influence pathogen-plant interactions in stable disease suppression. PMID:24699870

Control structures for high speed processors

NASA Technical Reports Server (NTRS)

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

1982-01-01

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

Strategic and Operational Plan for Integrating Transcriptomics ...

EPA Pesticide Factsheets

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

High-Throughput Experimental Approach Capabilities | Materials Science |

Science.gov Websites

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

Plaque bacterial microbiome diversity in children younger than 30 months with or without caries prior to eruption of second primary molars.

PubMed

Xu, He; Hao, Wenjing; Zhou, Qiong; Wang, Wenhong; Xia, Zhongkui; Liu, Chuan; Chen, Xiaochi; Qin, Man; Chen, Feng

2014-01-01

Our primary objective is to phylogenetically characterize the supragingival plaque bacterial microbiome of children prior to eruption of second primary molars by pyrosequencing method for studying etiology of early childhood caries. Supragingival plaque samples were collected from 10 caries children and 9 caries-free children. Plaque DNA was extracted, used to generate DNA amplicons of the V1-V3 hypervariable region of the bacterial 16S rRNA gene, and subjected to 454-pyrosequencing. On average, over 22,000 sequences per sample were generated. High bacterial diversity was noted in the plaque of children with caries [170 operational taxonomical units (OTU) at 3% divergence] and caries-free children (201 OTU at 3% divergence) with no significant difference. A total of 8 phyla, 15 classes, 21 orders, 30 families, 41 genera and 99 species were represented. In addition, five predominant phyla (Firmicute, Fusobacteria, Proteobacteria, Bacteroidetes and Actinobacteria) and seven genera (Leptotrichia, Streptococcus, Actinomyces, Prevotella, Porphyromonas, Neisseria, and Veillonella) constituted a majority of contents of the total microbiota, independent of the presence or absence of caries. Principal Component Analysis (PCA) presented that caries-related genera included Streptococcus and Veillonella; while Leptotrichia, Selenomonas, Fusobacterium, Capnocytophaga and Porphyromonas were more related to the caries-free samples. Neisseria and Prevotella presented approximately in between. In both groups, the degree of shared organism lineages (as defined by species-level OTUs) among individual supragingival plaque microbiomes was minimal. Our study represented for the first time using pyrosequencing to elucidate and monitor supragingival plaque bacterial diversity at such young age with second primary molar unerrupted. Distinctions were revealed between caries and caries-free microbiomes in terms of microbial community structure. We observed differences in abundance for several microbial groups between the caries and caries-free host populations, which were consistent with the ecological plaque hypothesis. Our approach and findings could be extended to correlating microbiomic changes after occlusion establishment and caries treatment.

Temporal Dynamics of Abundance and Composition of Nitrogen-Fixing Communities across Agricultural Soils

PubMed Central

Pereira e Silva, Michele C.; Schloter-Hai, Brigitte; Schloter, Michael; van Elsas, Jan Dirk; Salles, Joana Falcão

2013-01-01

Background Despite the fact that the fixation of nitrogen is one of the most significant nutrient processes in the terrestrial ecosystem, a thorough study of the spatial and temporal patterns in the abundance and distribution of N-fixing communities has been missing so far. Methodology/Principal Findings In order to understand the dynamics of diazotrophic communities and their resilience to external changes, we quantified the abundance and characterized the bacterial community structures based on the nifH gene, using real-time PCR, PCR-DGGE and 454-pyrosequencing, across four representative Dutch soils during one growing season. In general, higher nifH gene copy numbers were observed in soils with higher pH than in those with lower pH, but lower numbers were related to increased nitrate and ammonium levels. Results from nifH gene pyrosequencing confirmed the observed PCR-DGGE patterns, which indicated that the N fixers are highly dynamic across time, shifting around 60%. Forward selection on CCA analysis identified N availability as the main driver of these variations, as well as of the evenness of the communities, leading to very unequal communities. Moreover, deep sequencing of the nifH gene revealed that sandy soils (B and D) had the lowest percentage of shared OTUs across time, compared with clayey soils (G and K), indicating the presence of a community under constant change. Cosmopolitan nifH species (present throughout the season) were affiliated with Bradyrhizobium , Azospirillum and Methylocistis, whereas other species increased their abundances progressively over time, when appropriate conditions were met, as was notably the case for Paenibacilus and Burkholderia. Conclusions Our study provides the first in-depth pyrosequencing analysis of the N-fixing community at both spatial and temporal scales, providing insights into the cosmopolitan and specific portions of the nitrogen fixing bacterial communities in soil. PMID:24058578

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

PubMed Central

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

2010-01-01

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

Multiplex pyrosequencing assay using AdvISER-MH-PYRO algorithm: a case for rapid and cost-effective genotyping analysis of prostate cancer risk-associated SNPs.

PubMed

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 analysis. Using this combined multiplex approach also substantially reduced the production of waste material. These genotyping assays appear therefore to be biologically, economically and ecologically highly relevant, being worth to be integrated in genetic-based predictive strategies for better selecting patients at risk for prostate cancer. In addition, the same approach could now equally be transposed to other clinical/research applications relying on the computation of MGRS based on multi-SNP genotyping.

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

EPA Pesticide Factsheets

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

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

NASA Astrophysics Data System (ADS)

Safi, Asmahan A.

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

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

PubMed

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.

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

PubMed Central

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

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

PubMed

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.

SVS: data and knowledge integration in computational biology.

PubMed

Zycinski, Grzegorz; Barla, Annalisa; Verri, Alessandro

2011-01-01

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

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2015-08-01

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

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

PubMed Central

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

2018-01-01

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

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

PubMed

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

2016-08-08

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

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

PubMed Central

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

2015-01-01

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. PMID:26585833

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

PubMed

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.

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

NASA Astrophysics Data System (ADS)

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

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

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

NASA Astrophysics Data System (ADS)

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

2015-10-01

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

Challenges in NMR-based structural genomics

NASA Astrophysics Data System (ADS)

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

2005-05-01

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

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

PubMed

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

2015-07-10

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

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

PubMed

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

2014-12-11

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

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

PubMed

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

2016-02-21

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

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

PubMed

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

2012-02-01

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

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2017-12-01

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

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

NASA Astrophysics Data System (ADS)

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

2015-12-01

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

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

PubMed Central

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

2015-01-01

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

Analysis of Stomach and Gut Microbiomes of the Eastern Oyster (Crassostrea virginica) from Coastal Louisiana, USA

PubMed Central

King, Gary M.; Judd, Craig; Kuske, Cheryl R.; Smith, Conor

2012-01-01

We used high throughput pyrosequencing to characterize stomach and gut content microbiomes of Crassostrea virginica, the Easter oyster, obtained from two sites, one in Barataria Bay (Hackberry Bay) and the other in Terrebonne Bay (Lake Caillou), Louisiana, USA. Stomach microbiomes in oysters from Hackberry Bay were overwhelmingly dominated by Mollicutes most closely related to Mycoplasma; a more rich community dominated by Planctomyctes occurred in Lake Caillou oyster stomachs. Gut communities for oysters from both sites differed from stomach communities, and harbored a relatively diverse assemblage of phylotypes. Phylotypes most closely related to Shewanella and a Chloroflexi strain dominated the Lake Caillou and Hackberry Bay gut microbiota, respectively. While many members of the stomach and gut microbiomes appeared to be transients or opportunists, a putative core microbiome was identified based on phylotypes that occurred in all stomach or gut samples only. The putative core stomach microbiome comprised 5 OTUs in 3 phyla, while the putative core gut microbiome contained 44 OTUs in 12 phyla. These results collectively revealed novel microbial communities within the oyster digestive system, the functions of the oyster microbiome are largely unknown. A comparison of microbiomes from Louisiana oysters with bacterial communities reported for other marine invertebrates and fish indicated that molluscan microbiomes were more similar to each other than to microbiomes of polychaetes, decapods and fish. PMID:23251548

Alkyl polyglucose enhancing propionic acid enriched short-chain fatty acids production during anaerobic treatment of waste activated sludge and mechanisms.

PubMed

Luo, Jingyang; Feng, Leiyu; Chen, Yinguang; Sun, Han; Shen, Qiuting; Li, Xiang; Chen, Hong

2015-04-15

Adding alkyl polyglucose (APG) into an anaerobic treatment system of waste activated sludge (WAS) was reported to remarkably improve the production of short-chain fatty acids (SCFAs), especially propionic acid via simultaneously accelerating solubilization and hydrolysis, enhancing acidification, inhibiting methanogenesis and balancing carbon to nitrogen (C/N) ratio of substrate. Not only the production of SCFAs, especially propionic acid, was significantly improved by APG, but also the feasible operation time was shortened. The SCFAs yield at 0.3 g APG per gram of total suspended solids (TSS) within 4 d was 2988 ± 60 mg chemical oxygen demand (COD) per liter, much higher than that those from sole WAS or sole WAS plus sole APG. The corresponding yield of propionic acid was 1312 ± 25 mg COD/L, 7.9-fold of sole WAS. Mechanism investigation showed that during anaerobic treatment of WAS in the presence of APG both the solubilization and hydrolysis were accelerated and the acidification was enhanced, while the methanogenesis was inhibited. Moreover, the activities of key enzymes involved in WAS hydrolysis and acidification were improved through the adjustment of C/N ratio of substrates with APG. The abundance of microorganisms responsible for organic compounds hydrolysis and SCFAs production was also observed to be greatly enhanced with APG via 454 high-throughput pyrosequencing analysis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Analysis of Stomach and Gut Microbiomes of the Eastern Oyster (Crassostrea virginica) from Coastal Louisiana, USA

DOE PAGES

King, Gary M.; Judd, Craig; Kuske, Cheryl R.; ...

2012-12-12

In this paper, we used high throughput pyrosequencing to characterize stomach and gut content microbiomes of Crassostrea virginica, the Easter oyster, obtained from two sites, one in Barataria Bay (Hackberry Bay) and the other in Terrebonne Bay (Lake Caillou), Louisiana, USA. Stomach microbiomes in oysters from Hackberry Bay were overwhelmingly dominated by Mollicutes most closely related to Mycoplasma; a more rich community dominated by Planctomyctes occurred in Lake Caillou oyster stomachs. Gut communities for oysters from both sites differed from stomach communities, and harbored a relatively diverse assemblage of phylotypes. Phylotypes most closely related to Shewanella and a Chloroflexi strainmore » dominated the Lake Caillou and Hackberry Bay gut microbiota, respectively. While many members of the stomach and gut microbiomes appeared to be transients or opportunists, a putative core microbiome was identified based on phylotypes that occurred in all stomach or gut samples only. The putative core stomach microbiome comprised 5 OTUs in 3 phyla, while the putative core gut microbiome contained 44 OTUs in 12 phyla. These results collectively revealed novel microbial communities within the oyster digestive system, the functions of the oyster microbiome are largely unknown. Finally, a comparison of microbiomes from Louisiana oysters with bacterial communities reported for other marine invertebrates and fish indicated that molluscan microbiomes were more similar to each other than to microbiomes of polychaetes, decapods and fish.« less

Epigenetic Biomarker to Support Classification into Pluripotent and Non-Pluripotent Cells

NASA Astrophysics Data System (ADS)

Lenz, Michael; Goetzke, Roman; Schenk, Arne; Schubert, Claudia; Veeck, Jürgen; Hemeda, Hatim; Koschmieder, Steffen; Zenke, Martin; Schuppert, Andreas; Wagner, Wolfgang

2015-03-01

Quality control of human induced pluripotent stem cells (iPSCs) can be performed by several methods. These methods are usually relatively labor-intensive, difficult to standardize, or they do not facilitate reliable quantification. Here, we describe a biomarker to distinguish between pluripotent and non-pluripotent cells based on DNA methylation (DNAm) levels at only three specific CpG sites. Two of these CpG sites were selected by their discriminatory power in 258 DNAm profiles - they were either methylated in pluripotent or non-pluripotent cells. The difference between these two β-values provides an Epi-Pluri-Score that was validated on independent DNAm-datasets (264 pluripotent and 1,951 non-pluripotent samples) with 99.9% specificity and 98.9% sensitivity. This score was complemented by a third CpG within the gene POU5F1 (OCT4), which better demarcates early differentiation events. We established pyrosequencing assays for the three relevant CpG sites and thereby correctly classified DNA of 12 pluripotent cell lines and 31 non-pluripotent cell lines. Furthermore, DNAm changes at these three CpGs were tracked in the course of differentiation of iPSCs towards mesenchymal stromal cells. The Epi-Pluri-Score does not give information on lineage-specific differentiation potential, but it provides a simple, reliable, and robust biomarker to support high-throughput classification into either pluripotent or non-pluripotent cells.

Biological floating bed and bio-contact oxidation processes for landscape water treatment: simultaneous removal of Microcystis aeruginosa, TOC, nitrogen and phosphorus.

PubMed

Su, Jun Feng; Liang, Dong Hui; Fu, Le; Wei, Li; Ma, Min

2018-06-13

The aim of this study was to identify algicidal bacteria J25 against the Microcystis aeruginosa (90.14%), Chlorella (78.75%), Scenedesmus (not inhibited), and Oscillatoria (90.12%). Meanwhile, we evaluate the SOD activity and efficiency of denitrification characteristics with Acinetobacter sp. J25. A novel hybrid bioreactor combined biological floating bed with bio-contact oxidation (BFBO) was designed for treating the landscape water, and the average removal efficiencies of nitrate-N, ammonia-N, nitrite-N, TN, TP, TOC, and algal cells were 91.14, 50, 87.86, 88.83, 33.07, 53.95, and 53.43%, respectively. A 454-pyrosequencing technology was employed to investigate the microbial communities of the BFBO reactor samples. The results showed that Acinetobacter sp. J25 was the dominant contributor for effective removal of N, algal cells, and TOC in the BFBO reactor. And the relative abundance of Acinetobacter showed increase trend with the delay of reaction time. Graphical abstract Biological floating bed and bio-contact oxidation (BFBO) as a novel hybrid bioreactor designed for simultaneous removal Microcystis aeruginosa, TOC, nitrogen, and phosphorus. And high-throughput sequencing data demonstrated that Acinetobacter sp. J25 was the dominate species in the reactor and played key roles in the removal of N, TOC, and M. aeruginosa. Proposed reaction mechanism of the BFBO.

Potential Accumulative Effect of the Herbicide Glyphosate on Glyphosate-Tolerant Maize Rhizobacterial Communities over a Three-Year Cultivation Period

PubMed Central

Barriuso, Jorge; Marín, Silvia; Mellado, Rafael P.

2011-01-01

Background Glyphosate is a herbicide that is liable to be used in the extensive cultivation of glyphosate-tolerant cultivars. The potential accumulation of the relative effect of glyphosate on the rhizobacterial communities of glyphosate-tolerant maize has been monitored over a period of three years. Methodology/Principal Findings The composition of rhizobacterial communities is known to vary with soil texture, hence, the analyses have been performed in two agricultural fields with a different soil texture. The accumulative effects of glyphosate have been monitored by means of high throughput DNA pyrosequencing of the bacterial DNA coding for the 16S rRNA hypervariable V6 region from rhizobacterial communities. The relative composition of the rhizobacterial communities does vary in each field over the three-year period. The overall distribution of the bacterial phyla seems to change from one year to the next similarly in the untreated and glyphosate-treated soils in both fields. The two methods used to estimate bacterial diversity offered consistent results and are equally suitable for diversity assessment. Conclusions/Significance The glyphosate treatment during the three-year period of seasonal cultivation in two different fields did not seem to significantly change the maize rhizobacterial communities when compared to those of the untreated soil. This may be particularly relevant with respect to a potential authorisation to cultivate glyphosate-tolerant maize in the European Union. PMID:22096595

Effect of broad- and narrow-spectrum antimicrobials on Clostridium difficile and microbial diversity in a model of the distal colon

PubMed Central

Rea, Mary C.; Dobson, Alleson; O'Sullivan, Orla; Crispie, Fiona; Fouhy, Fiona; Cotter, Paul D.; Shanahan, Fergus; Kiely, Barry; Hill, Colin; Ross, R. Paul

2011-01-01

Vancomycin, metronidazole, and the bacteriocin lacticin 3147 are active against a wide range of bacterial species, including Clostridium difficile. We demonstrate that, in a human distal colon model, the addition of each of the three antimicrobials resulted in a significant decrease in numbers of C. difficile. However, their therapeutic use in the gastrointestinal tract may be compromised by their broad spectrum of activity, which would be expected to significantly impact on other members of the human gut microbiota. We used high-throughput pyrosequencing to compare the effect of each antimicrobial on the composition of the microbiota. All three treatments resulted in a decrease in the proportion of sequences assigned to the phyla Firmicutes and Bacteroidetes, with a corresponding increase in those assigned to members of the Proteobacteria. One possible means of avoiding such “collateral damage” would involve the application of a narrow-spectrum antimicrobial with specific anti-C. difficile activity. We tested this hypothesis using thuricin CD, a narrow-spectrum bacteriocin produced by Bacillus thuringiensis, which is active against C. difficile. The results demonstrated that this bacteriocin was equally effective at killing C. difficile in the distal colon model but had no significant impact on the composition of the microbiota. This offers the possibility of developing a targeted approach to eliminating C. difficile in the colon, without collateral damage. PMID:20616009

Aggregating Data for Computational Toxicology Applications ...

EPA Pesticide Factsheets

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

SUBCELLULAR PHARMACOKINETICS AND ITS POTENTIAL FOR LIBRARY FOCUSING (R826652)

EPA Science Inventory

Abstract

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

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

EPA Science Inventory

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

High-Throughput Analysis and Automation for Glycomics Studies.

PubMed

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

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

Microbiota and epigenetic regulation of inflammatory mediators in type 2 diabetes and obesity.

PubMed

Remely, M; Aumueller, E; Jahn, D; Hippe, B; Brath, H; Haslberger, A G

2014-03-01

Metabolic syndrome is associated with alterations in the structure of the gut microbiota leading to low-grade inflammatory responses. An increased penetration of the impaired gut membrane by bacterial components is believed to induce this inflammation, possibly involving epigenetic alteration of inflammatory molecules such as Toll-like receptors (TLRs). We evaluated changes of the gut microbiota and epigenetic DNA methylation of TLR2 and TLR4 in three groups of subjects: type 2 diabetics under glucagon-like peptide-1 agonist therapy, obese individuals without established insulin resistance, and a lean control group. Clostridium cluster IV, Clostridium cluster XIVa, lactic acid bacteria, Faecalibacterium prausnitzii and Bacteroidetes abundances were analysed by PCR and 454 high-throughput sequencing. The epigenetic methylation in the regulatory region of TLR4 and TLR2 was analysed using bisulfite conversion and pyrosequencing. We observed a significantly higher ratio of Firmicutes/ Bacteroidetes in type 2 diabetics compared to lean controls and obese. Major differences were shown in lactic acid bacteria, with the highest abundance in type 2 diabetics, followed by obese and lean participants. In comparison, F. prausnitzii was least abundant in type 2 diabetics, and most abundant in lean controls. Methylation analysis of four CpGs in the first exon of TLR4 showed significantly lower methylation in obese individuals, but no significant difference between type 2 diabetics and lean controls. Methylation of seven CpGs in the promoter region of TLR2 was significantly lower in type 2 diabetics compared to obese subjects and lean controls. The methylation levels of both TLRs were significantly correlated with body mass index. Our data suggest that changes in gut microbiota and thus cell wall components are involved in the epigenetic regulation of inflammatory reactions. An improved diet targeted to induce gut microbial balance and in the following even epigenetic changes of pro-inflammatory genes may be effective in the prevention of metabolic syndrome.

Lyssavirus Detection and Typing Using Pyrosequencing▿#‖

PubMed Central

De Benedictis, Paola; De Battisti, Cristian; Dacheux, Laurent; Marciano, Sabrina; Ormelli, Silvia; Salomoni, Angela; Caenazzo, Silvia Tiozzo; Lepelletier, Anthony; Bourhy, Hervé; Capua, Ilaria; Cattoli, Giovanni

2011-01-01

Rabies is a fatal zoonosis caused by a nonsegmented negative-strand RNA virus, namely, rabies virus (RABV). Apart from RABV, at least 10 additional species are known as rabies-related lyssaviruses (RRVs), and some of them are responsible for occasional spillovers into humans. More lyssaviruses have also been detected recently in different bat ecosystems, thanks to the application of molecular diagnostic methods. Due to the variety of the members of the genus Lyssavirus, there is the necessity to develop a reliable molecular assay for rabies diagnosis able to detect and differentiate among the existing rabies and rabies-related viruses. In the present study, a pyrosequencing protocol targeting the 3′ terminus of the nucleoprotein (N) gene was applied for the rapid characterization of lyssaviruses. Correct identification of species was achieved for each sample tested. Results from the pyrosequencing assay were also confirmed by those obtained using the Sanger sequencing method. A pan-lyssavirus one-step reverse transcription (RT)-PCR was developed within the framework of the pyrosequencing procedure. The sensitivity (Se) of the one-step RT-PCR assay was determined by using in vitro-transcribed RNA and serial dilutions of titrated viruses. The assay demonstrated high analytical and relative specificity (Sp) (98.94%) and sensitivity (99.71%). To date, this is the first case in which pyrosequencing has been applied for lyssavirus identification using a cheaper diagnostic approach than the one for all the other protocols for rapid typing that we are acquainted with. Results from this study indicate that this procedure is suitable for lyssavirus detection in samples of both human and animal origin. PMID:21389152

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

NASA Astrophysics Data System (ADS)

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

2017-12-01

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

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

PubMed

Xiang, X D

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

Predicting Novel Bulk Metallic Glasses via High- Throughput Calculations

NASA Astrophysics Data System (ADS)

Perim, E.; Lee, D.; Liu, Y.; Toher, C.; Gong, P.; Li, Y.; Simmons, W. N.; Levy, O.; Vlassak, J.; Schroers, J.; Curtarolo, S.

Bulk metallic glasses (BMGs) are materials which may combine key properties from crystalline metals, such as high hardness, with others typically presented by plastics, such as easy processability. However, the cost of the known BMGs poses a significant obstacle for the development of applications, which has lead to a long search for novel, economically viable, BMGs. The emergence of high-throughput DFT calculations, such as the library provided by the AFLOWLIB consortium, has provided new tools for materials discovery. We have used this data to develop a new glass forming descriptor combining structural factors with thermodynamics in order to quickly screen through a large number of alloy systems in the AFLOWLIB database, identifying the most promising systems and the optimal compositions for glass formation. National Science Foundation (DMR-1436151, DMR-1435820, DMR-1436268).

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

PubMed

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

2016-09-01

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

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2005-12-01

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

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

PubMed Central

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

2005-01-01

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

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

NASA Astrophysics Data System (ADS)

Mughal, A.; Newman, H.

2017-10-01

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

DNA methylation and hydroxymethylation analyses of the active LINE-1 subfamilies in mice.

PubMed

Murata, Yui; Bundo, Miki; Ueda, Junko; Kubota-Sakashita, Mie; Kasai, Kiyoto; Kato, Tadafumi; Iwamoto, Kazuya

2017-10-19

Retrotransposon long interspersed nuclear element-1 (LINE-1) occupies a large proportion of the mammalian genome, comprising approximately 100,000 genomic copies in mice. Epigenetic status of the 5' untranslated region (5'-UTR) of LINE-1 is critical for its promoter activity. DNA methylation levels in the 5'-UTR of human active LINE-1 subfamily can be measured by well-established methods, such as a pyrosequencing-based assay. However, because of the considerable sequence and structural diversity in LINE-1 among species, methods for such assays should be adapted for the species of interest. Here we developed pyrosequencing-based assays to examine methylcytosine (mC) and hydroxymethylcytosine (hmC) levels of the three active LINE-1 subfamilies in mice (TfI, A, and GfII). Using these assays, we quantified mC and hmC levels in four brain regions and four nonbrain tissues including tail, heart, testis, and ovary. We observed tissue- and subfamily-specific mC and hmC differences. We also found that mC levels were strongly correlated among different brain regions, but mC levels of the testis showed a poor correlation with those of other tissues. Interestingly, mC levels in the A and GfII subfamilies were highly correlated, possibly reflecting their close evolutionary relationship. Our assays will be useful for exploring the epigenetic regulation of the active LINE-1 subfamilies in mice.

Time-Resolved DNA Stable Isotope Probing Links Desulfobacterales- and Coriobacteriaceae-Related Bacteria to Anaerobic Degradation of Benzene under Methanogenic Conditions

PubMed Central

Noguchi, Mana; Kurisu, Futoshi; Kasuga, Ikuro; Furumai, Hiroaki

2014-01-01

To identify the microorganisms involved in benzene degradation, DNA-stable isotope probing (SIP) with 13C-benzene was applied to a methanogenic benzene-degrading enrichment culture. Pyrosequencing of ribosomal RNA (rRNA) gene sequences revealed that the community structure was highly complex in spite of a 3-year incubation only with benzene. The culture degraded 98% of approximately 1 mM 13C-benzene and mineralized 72% of that within 63 d. The terminal restriction fragment length polymorphism (T-RFLP) profiles of the buoyant density fractions revealed the incorporation of 13C into two phylotypes after 64 d. These two phylotypes were determined to be Desulfobacterales- and Coriobacteriaceae-related bacteria by cloning and sequencing of the 16S rRNA gene in the 13C-labeled DNA abundant fraction. Comparative pyrosequencing analysis of the buoyant density fractions of 12C- and 13C-labeled samples indicated the incorporation of 13C into three bacterial and one archaeal OTUs related to Desulfobacterales, Coriobacteriales, Rhodocyclaceae, and Methanosarcinales. The first two OTUs included the bacteria detected by T-RFLP-cloning-sequencing analysis. Furthermore, time-resolved SIP analysis confirmed that the activity of all these microbes appeared at the earliest stage of degradation. In this methanogenic culture, Desulfobacterales- and Coriobacteriaceae-related bacteria were most likely to be the major benzene degraders. PMID:24909708

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

PubMed Central

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

2015-01-01

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

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

EPA Pesticide Factsheets

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

New Toxico-Cheminformatics & Computational Toxicology ...

EPA Pesticide Factsheets

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

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

PubMed

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

2016-07-03

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

Predicting organ toxicity using in vitro bioactivity data and chemical structure

EPA Science Inventory

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

Pyrosequencing analysis of microbial communities in hollow fiber-membrane biofilm reactors system for treating high-strength nitrogen wastewater.

PubMed

Park, Jung-Hun; Choi, Okkyoung; Lee, Tae-Ho; Kim, Hyunook; Sang, Byoung-In

2016-11-01

Wastewaters from swine farms, nitrogen-dealing industries or side-stream processes of a wastewater treatment plant (e.g., anaerobic digesters, sludge thickening processes, etc.) are characterized by low C/N ratios and not easily treatable. In this study, a hollow fiber-membrane biofilm reactors (HF-MBfR) system consisting of an O2-based HF-MBfR and an H2-based HF-MBfR was applied for treating high-strength wastewater. The reactors were continuously operated with low supply of O2 and H2 and without any supply of organic carbon for 250 d. Gradual increase of ammonium and nitrate concentration in the influent showed stable and high nitrogen removal efficiency, and the maximum ammonium and nitrate removal rates were 0.48 kg NH4(+)-N m(-3) d(-1) and 0.55 kg NO3(-)-N m(-3) d(-1), respectively. The analysis of the microbial communities using pyrosequencing analysis indicated that Nitrosospira multiformis, ammonium-oxidizing bacteria, and Nitrobacter winogradskyi and Nitrobacter vulgaris, nitrite-oxidizing bacteria were highly enriched in the O2-based HF-MBfR. In the H2-based HF-MBfR, hydrogenotrophic denitrifying bacteria belonging to the family of Thiobacillus and Comamonadaceae were initially dominant, but were replaced to heterotrophic denitrifiers belonging to Rhodocyclaceae and Rhodobacteraceae utilizing by-products induced from autotrophic denitrifying bacteria. The pyrosequencing analysis of microbial communities indicates that the autotrophic HF-MBfRs system well developed autotrophic nitrifying and denitrifying bacteria within a relatively short period to accomplish almost complete nitrogen removal. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

EPA Pesticide Factsheets

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

Facile fabrication of nanofluidic diode membranes using anodic aluminium oxide

NASA Astrophysics Data System (ADS)

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

2012-08-01

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

Neutron reflecting supermirror structure

DOEpatents

Wood, James L.

1992-01-01

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

5'-Serial Analysis of Gene Expression studies reveal a transcriptomic switch during fruiting body development in Coprinopsis cinerea

PubMed Central

2013-01-01

Background The transition from the vegetative mycelium to the primordium during fruiting body development is the most complex and critical developmental event in the life cycle of many basidiomycete fungi. Understanding the molecular mechanisms underlying this process has long been a goal of research on basidiomycetes. Large scale assessment of the expressed transcriptomes of these developmental stages will facilitate the generation of a more comprehensive picture of the mushroom fruiting process. In this study, we coupled 5'-Serial Analysis of Gene Expression (5'-SAGE) to high-throughput pyrosequencing from 454 Life Sciences to analyze the transcriptomes and identify up-regulated genes among vegetative mycelium (Myc) and stage 1 primordium (S1-Pri) of Coprinopsis cinerea during fruiting body development. Results We evaluated the expression of >3,000 genes in the two respective growth stages and discovered that almost one-third of these genes were preferentially expressed in either stage. This identified a significant turnover of the transcriptome during the course of fruiting body development. Additionally, we annotated more than 79,000 transcription start sites (TSSs) based on the transcriptomes of the mycelium and stage 1 primoridum stages. Patterns of enrichment based on gene annotations from the GO and KEGG databases indicated that various structural and functional protein families were uniquely employed in either stage and that during primordial growth, cellular metabolism is highly up-regulated. Various signaling pathways such as the cAMP-PKA, MAPK and TOR pathways were also identified as up-regulated, consistent with the model that sensing of nutrient levels and the environment are important in this developmental transition. More than 100 up-regulated genes were also found to be unique to mushroom forming basidiomycetes, highlighting the novelty of fruiting body development in the fungal kingdom. Conclusions We implicated a wealth of new candidate genes important to early stages of mushroom fruiting development, though their precise molecular functions and biological roles are not yet fully known. This study serves to advance our understanding of the molecular mechanisms of fruiting body development in the model mushroom C. cinerea. PMID:23514374

Pyrosequencing Analysis of Bench-Scale Nitrifying BiofiltersRemoving Trihalomethanes

EPA Science Inventory

The bacterial biofilm communities in four nitrifying biofilters degrading regulated drinking water trihalomethanes were characterized by 454 pyrosequencing. The three most abundant phylotypes based on total diversity were Nitrosomonas (70%), Nitrobacter (14%), and Chitinophagace...

Changes in N-Transforming Archaea and Bacteria in Soil during the Establishment of Bioenergy Crops

PubMed Central

Mao, Yuejian; Yannarell, Anthony C.; Mackie, Roderick I.

2011-01-01

Widespread adaptation of biomass production for bioenergy may influence important biogeochemical functions in the landscape, which are mainly carried out by soil microbes. Here we explore the impact of four potential bioenergy feedstock crops (maize, switchgrass, Miscanthus X giganteus, and mixed tallgrass prairie) on nitrogen cycling microorganisms in the soil by monitoring the changes in the quantity (real-time PCR) and diversity (barcoded pyrosequencing) of key functional genes (nifH, bacterial/archaeal amoA and nosZ) and 16S rRNA genes over two years after bioenergy crop establishment. The quantities of these N-cycling genes were relatively stable in all four crops, except maize (the only fertilized crop), in which the population size of AOB doubled in less than 3 months. The nitrification rate was significantly correlated with the quantity of ammonia-oxidizing archaea (AOA) not bacteria (AOB), indicating that archaea were the major ammonia oxidizers. Deep sequencing revealed high diversity of nifH, archaeal amoA, bacterial amoA, nosZ and 16S rRNA genes, with 229, 309, 330, 331 and 8989 OTUs observed, respectively. Rarefaction analysis revealed the diversity of archaeal amoA in maize markedly decreased in the second year. Ordination analysis of T-RFLP and pyrosequencing results showed that the N-transforming microbial community structures in the soil under these crops gradually differentiated. Thus far, our two-year study has shown that specific N-transforming microbial communities develop in the soil in response to planting different bioenergy crops, and each functional group responded in a different way. Our results also suggest that cultivation of maize with N-fertilization increases the abundance of AOB and denitrifiers, reduces the diversity of AOA, and results in significant changes in the structure of denitrification community. PMID:21935454

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

PubMed

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

2015-10-01

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

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

PubMed Central

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

2015-01-01

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

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

EPA Science Inventory

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

Higher Throughput Calorimetry: Opportunities, Approaches and Challenges

PubMed Central

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

2010-01-01

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

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

PubMed

Zimmerlin, Alfred; Kiffe, Michael

2013-01-01

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

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

PubMed Central

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

2016-01-01

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

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

PubMed Central

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

2014-01-01

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

Identification of candidate genes for drought tolerance in coffee by high-throughput sequencing in the shoot apex of different Coffea arabica cultivars.

PubMed

Mofatto, Luciana Souto; Carneiro, Fernanda de Araújo; Vieira, Natalia Gomes; Duarte, Karoline Estefani; Vidal, Ramon Oliveira; Alekcevetch, Jean Carlos; Cotta, Michelle Guitton; Verdeil, Jean-Luc; Lapeyre-Montes, Fabienne; Lartaud, Marc; Leroy, Thierry; De Bellis, Fabien; Pot, David; Rodrigues, Gustavo Costa; Carazzolle, Marcelo Falsarella; Pereira, Gonçalo Amarante Guimarães; Andrade, Alan Carvalho; Marraccini, Pierre

2016-04-19

Drought is a widespread limiting factor in coffee plants. It affects plant development, fruit production, bean development and consequently beverage quality. Genetic diversity for drought tolerance exists within the coffee genus. However, the molecular mechanisms underlying the adaptation of coffee plants to drought are largely unknown. In this study, we compared the molecular responses to drought in two commercial cultivars (IAPAR59, drought-tolerant and Rubi, drought-susceptible) of Coffea arabica grown in the field under control (irrigation) and drought conditions using the pyrosequencing of RNA extracted from shoot apices and analysing the expression of 38 candidate genes. Pyrosequencing from shoot apices generated a total of 34.7 Mbp and 535,544 reads enabling the identification of 43,087 clusters (41,512 contigs and 1,575 singletons). These data included 17,719 clusters (16,238 contigs and 1,575 singletons) exclusively from 454 sequencing reads, along with 25,368 hybrid clusters assembled with 454 sequences. The comparison of DNA libraries identified new candidate genes (n = 20) presenting differential expression between IAPAR59 and Rubi and/or drought conditions. Their expression was monitored in plagiotropic buds, together with those of other (n = 18) candidates genes. Under drought conditions, up-regulated expression was observed in IAPAR59 but not in Rubi for CaSTK1 (protein kinase), CaSAMT1 (SAM-dependent methyltransferase), CaSLP1 (plant development) and CaMAS1 (ABA biosynthesis). Interestingly, the expression of lipid-transfer protein (nsLTP) genes was also highly up-regulated under drought conditions in IAPAR59. This may have been related to the thicker cuticle observed on the abaxial leaf surface in IAPAR59 compared to Rubi. The full transcriptome assembly of C. arabica, followed by functional annotation, enabled us to identify differentially expressed genes related to drought conditions. Using these data, candidate genes were selected and their differential expression profiles were confirmed by qPCR experiments in plagiotropic buds of IAPAR59 and Rubi under drought conditions. As regards the genes up-regulated under drought conditions, specifically in the drought-tolerant IAPAR59, several corresponded to orphan genes but also to genes coding proteins involved in signal transduction pathways, as well as ABA and lipid metabolism, for example. The identification of these genes should help advance our understanding of the genetic determinism of drought tolerance in coffee.

Performance of Different Analytical Software Packages in Quantification of DNA Methylation by Pyrosequencing.

PubMed

Grasso, Chiara; Trevisan, Morena; Fiano, Valentina; Tarallo, Valentina; De Marco, Laura; Sacerdote, Carlotta; Richiardi, Lorenzo; Merletti, Franco; Gillio-Tos, Anna

2016-01-01

Pyrosequencing has emerged as an alternative method of nucleic acid sequencing, well suited for many applications which aim to characterize single nucleotide polymorphisms, mutations, microbial types and CpG methylation in the target DNA. The commercially available pyrosequencing systems can harbor two different types of software which allow analysis in AQ or CpG mode, respectively, both widely employed for DNA methylation analysis. Aim of the study was to assess the performance for DNA methylation analysis at CpG sites of the two pyrosequencing software which allow analysis in AQ or CpG mode, respectively. Despite CpG mode having been specifically generated for CpG methylation quantification, many investigations on this topic have been carried out with AQ mode. As proof of equivalent performance of the two software for this type of analysis is not available, the focus of this paper was to evaluate if the two modes currently used for CpG methylation assessment by pyrosequencing may give overlapping results. We compared the performance of the two software in quantifying DNA methylation in the promoter of selected genes (GSTP1, MGMT, LINE-1) by testing two case series which include DNA from paraffin embedded prostate cancer tissues (PC study, N = 36) and DNA from blood fractions of healthy people (DD study, N = 28), respectively. We found discrepancy in the two pyrosequencing software-based quality assignment of DNA methylation assays. Compared to the software for analysis in the AQ mode, less permissive criteria are supported by the Pyro Q-CpG software, which enables analysis in CpG mode. CpG mode warns the operators about potential unsatisfactory performance of the assay and ensures a more accurate quantitative evaluation of DNA methylation at CpG sites. The implementation of CpG mode is strongly advisable in order to improve the reliability of the methylation analysis results achievable by pyrosequencing.

Multiplex pyrosequencing method to determine CYP2C9*3, VKORC1*2, and CYP4F2*3 polymorphisms simultaneously: its application to a Korean population and comparisons with other ethnic groups.

PubMed

Kim, Kyoung-Ah; Song, Wan-Geun; Lee, Hae-Mi; Joo, Hyun-Jin; Park, Ji-Young

2014-11-01

Warfarin is an anticoagulant that is difficult to administer because of the wide variation in dose requirements to achieve a therapeutic effect. CYP2C9, VKROC1, and CYP4F2 play important roles in warfarin metabolism, and their genetic polymorphisms are related to the variability in dose determination. In this study we describe a new multiplex pyrosequencing method to identify CYP2C9*3 (rs1057910), VKORC1*2 (rs9923231), and CYP4F2*3 (rs2108661) simultaneously. A multiplex pyrosequencing method to simultaneously detect CYP2C9*3, VKORC1*2, and CYP4F2*3 alleles was designed. We assessed the allele frequencies of the polymorphisms in 250 Korean subjects using the multiplex pyrosequencing method. The results showed 100 % concordance between single and multiplex pyrosequencing methods, and the polymorphisms identified by pyrosequencing were also validated with the direct sequencing method. The allele frequencies of these polymorphisms in this population were as follows: 0.040 for CYP2C9*3, 0.918 for VKORC1*2, and 0.416 for CYP4F2*3. Although the allele frequencies of the CYP2C9*3 and VKROC1*2 were comparable to those in Japanese and Chinese populations, their frequencies in this Korean population differed from those in other ethnic groups; the CYP4F2*3 frequency was the highest among other ethnic populations including Chinese and Japanese populations. The pyrosequencing methods developed were rapid and reliable for detecting CYP2C9*3, VKORC1*2, and CYP4F2*3. Large ethnic differences in the frequency of these genetic polymorphisms were noted among ethnic groups. CYP4F2*3 exhibited its highest allele frequency among other ethnic populations compared to that in a Korean population.

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

DTIC Science & Technology

2015-05-26

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

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

PubMed

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

2013-08-01

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

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

PubMed Central

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

2013-01-01

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

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

EPA Science Inventory

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

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

Federal Register 2010, 2011, 2012, 2013, 2014

2010-03-04

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

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

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-07

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

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

PubMed

Hayden, Eric J

2016-08-15

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

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

PubMed

Hickey, Scott F; Hammond, Ming C

2014-03-20

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

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

PubMed

Thielmann, Yvonne; Koepke, Juergen; Michel, Hartmut

2012-06-01

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

High-Throughput Characterization of Porous Materials Using Graphics Processing Units

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

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

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