Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification.

PubMed

Ziesemer, Kirsten A; Mann, Allison E; Sankaranarayanan, Krithivasan; Schroeder, Hannes; Ozga, Andrew T; Brandt, Bernd W; Zaura, Egija; Waters-Rist, Andrea; Hoogland, Menno; Salazar-García, Domingo C; Aldenderfer, Mark; Speller, Camilla; Hendy, Jessica; Weston, Darlene A; MacDonald, Sandy J; Thomas, Gavin H; Collins, Matthew J; Lewis, Cecil M; Hofman, Corinne; Warinner, Christina

2015-11-13

To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341-534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions.

Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification

PubMed Central

Ziesemer, Kirsten A.; Mann, Allison E.; Sankaranarayanan, Krithivasan; Schroeder, Hannes; Ozga, Andrew T.; Brandt, Bernd W.; Zaura, Egija; Waters-Rist, Andrea; Hoogland, Menno; Salazar-García, Domingo C.; Aldenderfer, Mark; Speller, Camilla; Hendy, Jessica; Weston, Darlene A.; MacDonald, Sandy J.; Thomas, Gavin H.; Collins, Matthew J.; Lewis, Cecil M.; Hofman, Corinne; Warinner, Christina

2015-01-01

To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341–534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions. PMID:26563586

Elucidation of taste- and odor-producing bacteria and toxigenic cyanobacteria in a Midwestern drinking water supply reservoir by shotgun metagenomics analysis

USGS Publications Warehouse

Otten, Timothy; Graham, Jennifer L.; Harris, Theodore D.; Dreher, Theo

2016-01-01

While commonplace in clinical settings, DNA-based assays for identification or enumeration of drinking water pathogens and other biological contaminants remain widely unadopted by the monitoring community. In this study, shotgun metagenomics was used to identify taste-and-odor producers and toxin-producing cyanobacteria over a 2-year period in a drinking water reservoir. The sequencing data implicated several cyanobacteria, including Anabaena spp.,Microcystis spp., and an unresolved member of the order Oscillatoriales as the likely principal producers of geosmin, microcystin, and 2-methylisoborneol (MIB), respectively. To further demonstrate this, quantitative PCR (qPCR) assays targeting geosmin-producing Anabaena and microcystin-producing Microcystis were utilized, and these data were fitted using generalized linear models and compared with routine monitoring data, including microscopic cell counts, sonde-based physicochemical analyses, and assays of all inorganic and organic nitrogen and phosphorus forms and fractions. The qPCR assays explained the greatest variation in observed geosmin (adjusted R2 = 0.71) and microcystin (adjusted R2 = 0.84) concentrations over the study period, highlighting their potential for routine monitoring applications. The origin of the monoterpene cyclase required for MIB biosynthesis was putatively linked to a periphytic cyanobacterial mat attached to the concrete drinking water inflow structure. We conclude that shotgun metagenomics can be used to identify microbial agents involved in water quality deterioration and to guide PCR assay selection or design for routine monitoring purposes. Finally, we offer estimates of microbial diversity and metagenomic coverage of our data sets for reference to others wishing to apply shotgun metagenomics to other lacustrine systems.

Shotgun proteomic monitoring of Clostridium acetobutylicum during stationary phase of butanol fermentation using xylose and comparison with the exponential phase

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

Sivagnanam, Kumaran; Raghavan, Vijaya G. S.; Shah, Manesh B

2012-01-01

Economically viable production of solvents through acetone butanol ethanol (ABE) fermentation requires a detailed understanding of Clostridium acetobutylicum. This study focuses on the proteomic profiling of C. acetobutylicum ATCC 824 from the stationary phase of ABE fermentation using xylose and compares with the exponential growth by shotgun proteomics approach. Comparative proteomic analysis revealed 22.9% of the C. acetobutylicum genome and 18.6% was found to be common in both exponential and stationary phases. The proteomic profile of C. acetobutylicum changed during the ABE fermentation such that 17 proteins were significantly differentially expressed between the two phases. Specifically, the expression of fivemore » proteins namely, CAC2873, CAP0164, CAP0165, CAC3298, and CAC1742 involved in the solvent production pathway were found to be significantly lower in the stationary phase compared to the exponential growth. Similarly, the expression of fucose isomerase (CAC2610), xylulose kinase (CAC2612), and a putative uncharacterized protein (CAC2611) involved in the xylose utilization pathway were also significantly lower in the stationary phase. These findings provide an insight into the metabolic behavior of C. acetobutylicum between different phases of ABE fermentation using xylose.« less

Proteomic analysis of the dorsal and ventral hippocampus of rats maintained on a high fat and refined sugar diet.

PubMed

Francis, Heather M; Mirzaei, Mehdi; Pardey, Margery C; Haynes, Paul A; Cornish, Jennifer L

2013-10-01

The typical Western diet, rich in high saturated fat and refined sugar (HFS), has been shown to increase cognitive decline with aging and Alzheimer's disease, and to affect cognitive functions that are dependent on the hippocampus, including memory processes and reversal learning. To investigate neurophysiological changes underlying these impairments, we employed a proteomic approach to identify differentially expressed proteins in the rat dorsal and ventral hippocampus following maintenance on an HFS diet. Rats maintained on the HFS diet for 8 weeks were impaired on a novel object recognition task that assesses memory and on a Morris Water Maze task assessing reversal learning. Quantitative label-free shotgun proteomic analysis was conducted on biological triplicates for each group. For the dorsal hippocampus, 59 proteins were upregulated and 36 downregulated in the HFS group compared to controls. Pathway ana-lysis revealed changes to proteins involved in molecular transport and cellular and molecular signaling, and changes to signaling pathways including calcium signaling, citrate cycle, and oxidative phosphorylation. For the ventral hippocampus, 25 proteins were upregulated and 27 downregulated in HFS fed rats. Differentially expressed proteins were involved in cell-to-cell signaling and interaction, and cellular and molecular function. Changes to signaling pathways included protein ubiquitination, ubiquinone biosynthesis, oxidative phosphorylation, and mitochondrial dysfunction. This is the first shotgun proteomics study to examine protein changes in the hippocampus following long-term consumption of a HFS diet, identifying changes to a large number of proteins including those involved in synaptic plasticity and energy metabolism. All MS data have been deposited in the ProteomeXchange with identifier PXD000028. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry.

PubMed

Ejsing, Christer S; Sampaio, Julio L; Surendranath, Vineeth; Duchoslav, Eva; Ekroos, Kim; Klemm, Robin W; Simons, Kai; Shevchenko, Andrej

2009-02-17

Although the transcriptome, proteome, and interactome of several eukaryotic model organisms have been described in detail, lipidomes remain relatively uncharacterized. Using Saccharomyces cerevisiae as an example, we demonstrate that automated shotgun lipidomics analysis enabled lipidome-wide absolute quantification of individual molecular lipid species by streamlined processing of a single sample of only 2 million yeast cells. By comparative lipidomics, we achieved the absolute quantification of 250 molecular lipid species covering 21 major lipid classes. This analysis provided approximately 95% coverage of the yeast lipidome achieved with 125-fold improvement in sensitivity compared with previous approaches. Comparative lipidomics demonstrated that growth temperature and defects in lipid biosynthesis induce ripple effects throughout the molecular composition of the yeast lipidome. This work serves as a resource for molecular characterization of eukaryotic lipidomes, and establishes shotgun lipidomics as a powerful platform for complementing biochemical studies and other systems-level approaches.

Soda pans of the Pannonian steppe harbor unique bacterial communities adapted to multiple extreme conditions.

PubMed

Szabó, Attila; Korponai, Kristóf; Kerepesi, Csaba; Somogyi, Boglárka; Vörös, Lajos; Bartha, Dániel; Márialigeti, Károly; Felföldi, Tamás

2017-05-01

Soda pans of the Pannonian steppe are unique environments regarding their physical and chemical characteristics: shallowness, high turbidity, intermittent character, alkaline pH, polyhumic organic carbon concentration, hypertrophic condition, moderately high salinity, sodium and carbonate ion dominance. The pans are highly productive environments with picophytoplankton predominance. Little is known about the planktonic bacterial communities inhabiting these aquatic habitats; therefore, amplicon sequencing and shotgun metagenomics were applied to reveal their composition and functional properties. Results showed a taxonomically complex bacterial community which was distinct from other soda lakes regarding its composition, e.g. the dominance of class Alphaproteobacteria was observed within phylum Proteobacteria. The shotgun metagenomic analysis revealed several functional gene components related to the harsh and at the same time hypertrophic environmental conditions, e.g. proteins involved in stress response, transport and hydrolase systems targeting phytoplankton-derived organic matter. This is the first detailed report on the indigenous planktonic bacterial communities coping with the multiple extreme conditions present in the unique soda pans of the Pannonian steppe.

Elucidation of Taste- and Odor-Producing Bacteria and Toxigenic Cyanobacteria in a Midwestern Drinking Water Supply Reservoir by Shotgun Metagenomic Analysis.

PubMed

Otten, Timothy G; Graham, Jennifer L; Harris, Theodore D; Dreher, Theo W

2016-09-01

While commonplace in clinical settings, DNA-based assays for identification or enumeration of drinking water pathogens and other biological contaminants remain widely unadopted by the monitoring community. In this study, shotgun metagenomics was used to identify taste-and-odor producers and toxin-producing cyanobacteria over a 2-year period in a drinking water reservoir. The sequencing data implicated several cyanobacteria, including Anabaena spp., Microcystis spp., and an unresolved member of the order Oscillatoriales as the likely principal producers of geosmin, microcystin, and 2-methylisoborneol (MIB), respectively. To further demonstrate this, quantitative PCR (qPCR) assays targeting geosmin-producing Anabaena and microcystin-producing Microcystis were utilized, and these data were fitted using generalized linear models and compared with routine monitoring data, including microscopic cell counts, sonde-based physicochemical analyses, and assays of all inorganic and organic nitrogen and phosphorus forms and fractions. The qPCR assays explained the greatest variation in observed geosmin (adjusted R(2) = 0.71) and microcystin (adjusted R(2) = 0.84) concentrations over the study period, highlighting their potential for routine monitoring applications. The origin of the monoterpene cyclase required for MIB biosynthesis was putatively linked to a periphytic cyanobacterial mat attached to the concrete drinking water inflow structure. We conclude that shotgun metagenomics can be used to identify microbial agents involved in water quality deterioration and to guide PCR assay selection or design for routine monitoring purposes. Finally, we offer estimates of microbial diversity and metagenomic coverage of our data sets for reference to others wishing to apply shotgun metagenomics to other lacustrine systems. Cyanobacterial toxins and microbial taste-and-odor compounds are a growing concern for drinking water utilities reliant upon surface water resources. Specific identification of the microorganism(s) responsible for water quality degradation is often complicated by the presence of co-occurring taxa capable of producing these undesirable metabolites. Here we present a framework for how shotgun metagenomics can be used to definitively identify problematic microorganisms and how these data can guide the development of rapid genetic assays for routine monitoring purposes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Elucidation of Taste- and Odor-Producing Bacteria and Toxigenic Cyanobacteria in a Midwestern Drinking Water Supply Reservoir by Shotgun Metagenomic Analysis

PubMed Central

Graham, Jennifer L.; Harris, Theodore D.

2016-01-01

ABSTRACT While commonplace in clinical settings, DNA-based assays for identification or enumeration of drinking water pathogens and other biological contaminants remain widely unadopted by the monitoring community. In this study, shotgun metagenomics was used to identify taste-and-odor producers and toxin-producing cyanobacteria over a 2-year period in a drinking water reservoir. The sequencing data implicated several cyanobacteria, including Anabaena spp., Microcystis spp., and an unresolved member of the order Oscillatoriales as the likely principal producers of geosmin, microcystin, and 2-methylisoborneol (MIB), respectively. To further demonstrate this, quantitative PCR (qPCR) assays targeting geosmin-producing Anabaena and microcystin-producing Microcystis were utilized, and these data were fitted using generalized linear models and compared with routine monitoring data, including microscopic cell counts, sonde-based physicochemical analyses, and assays of all inorganic and organic nitrogen and phosphorus forms and fractions. The qPCR assays explained the greatest variation in observed geosmin (adjusted R2 = 0.71) and microcystin (adjusted R2 = 0.84) concentrations over the study period, highlighting their potential for routine monitoring applications. The origin of the monoterpene cyclase required for MIB biosynthesis was putatively linked to a periphytic cyanobacterial mat attached to the concrete drinking water inflow structure. We conclude that shotgun metagenomics can be used to identify microbial agents involved in water quality deterioration and to guide PCR assay selection or design for routine monitoring purposes. Finally, we offer estimates of microbial diversity and metagenomic coverage of our data sets for reference to others wishing to apply shotgun metagenomics to other lacustrine systems. IMPORTANCE Cyanobacterial toxins and microbial taste-and-odor compounds are a growing concern for drinking water utilities reliant upon surface water resources. Specific identification of the microorganism(s) responsible for water quality degradation is often complicated by the presence of co-occurring taxa capable of producing these undesirable metabolites. Here we present a framework for how shotgun metagenomics can be used to definitively identify problematic microorganisms and how these data can guide the development of rapid genetic assays for routine monitoring purposes. PMID:27342564

DeMix Workflow for Efficient Identification of Cofragmented Peptides in High Resolution Data-dependent Tandem Mass Spectrometry*

PubMed Central

Zhang, Bo; Pirmoradian, Mohammad; Chernobrovkin, Alexey; Zubarev, Roman A.

2014-01-01

Based on conventional data-dependent acquisition strategy of shotgun proteomics, we present a new workflow DeMix, which significantly increases the efficiency of peptide identification for in-depth shotgun analysis of complex proteomes. Capitalizing on the high resolution and mass accuracy of Orbitrap-based tandem mass spectrometry, we developed a simple deconvolution method of “cloning” chimeric tandem spectra for cofragmented peptides. Additional to a database search, a simple rescoring scheme utilizes mass accuracy and converts the unwanted cofragmenting events into a surprising advantage of multiplexing. With the combination of cloning and rescoring, we obtained on average nine peptide-spectrum matches per second on a Q-Exactive workbench, whereas the actual MS/MS acquisition rate was close to seven spectra per second. This efficiency boost to 1.24 identified peptides per MS/MS spectrum enabled analysis of over 5000 human proteins in single-dimensional LC-MS/MS shotgun experiments with an only two-hour gradient. These findings suggest a change in the dominant “one MS/MS spectrum - one peptide” paradigm for data acquisition and analysis in shotgun data-dependent proteomics. DeMix also demonstrated higher robustness than conventional approaches in terms of lower variation among the results of consecutive LC-MS/MS runs. PMID:25100859

Shotgun proteomics approach to characterizing the embryonic proteome of the silkworm, Bombyx mori, at labrum appearance stage.

PubMed

Li, J-Y; Chen, X; Hosseini Moghaddam, S H; Chen, M; Wei, H; Zhong, B-X

2009-10-01

The shotgun approach has gained considerable acknowledgement in recent years as a dominant strategy in proteomics. We observed a dramatic increase of specific protein spots in two-dimensional electrophoresis (2-DE) gels of the silkworm (Bombyx mori) embryo at labrum appearance, a characteristic stage during embryonic development of silkworm which is involved with temperature increase by silkworm raiser. We employed shotgun liquid chromatography tandem mass spectrometry (LC-MS/MS) technology to analyse the proteome of B. mori embryos at this stage. A total of 2168 proteins were identified with an in-house database. Approximately 47% of them had isoelectric point (pI) values distributed theoretically in the range pI 5-7 and approximately 60% of them had molecular weights of 15-45 kDa. Furthermore, 111 proteins had an pI greater than 10 and were difficult to separate by 2-DE. Many important functional proteins related to embryonic development, stress response, DNA transcription/translation, cell growth, proliferation and differentiation, organogenesis and reproduction were identified. Among them proteins related to nervous system development were noticeable. All known heat shock proteins (HSPs) were detected in this developmental stage of B. mori embryo. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed energetic metabolism at this stage. These results were expected to provide more information for proteomic monitoring of the insect embryo and better understanding of the spatiotemporal expression of genes during embryonic developmental processes.

Profiling and relative quantification of phosphatidylethanolamine based on acetone stable isotope derivatization.

PubMed

Wang, Xiang; Wei, Fang; Xu, Ji-Qu; Lv, Xin; Dong, Xu-Yan; Han, Xianlin; Quek, Siew-Young; Huang, Feng-Hong; Chen, Hong

2016-01-01

Phosphatidylethanolamine (PE) is considered to be one of the pivotal lipids for normal cellular function as well as disease initiation and progression. In this study, a simple, efficient, reliable, and inexpensive method for the qualitative analysis and relative quantification of PE, based on acetone stable isotope derivatization combined with double neutral loss scan-shotgun electrospray ionization tandem-quadrupole mass spectrometry analysis (ASID-DNLS-Shotgun ESI-MS/MS), was developed. The ASID method led to alkylation of the primary amino groups of PE with an isopropyl moiety. The use of acetone (d0-acetone) and deuterium-labeled acetone (d6-acetone) introduced a 6 Da mass shift that was ideally suited for relative quantitative analysis, and enhanced sensitivity for mass analysis. The DNLS model was introduced to simultaneously analyze the differential derivatized PEs by shotgun ESI-MS/MS with high selectivity and accuracy. The reaction specificity, labeling efficiency, and linearity of the ASID method were thoroughly evaluated in this study. Its excellent applicability was validated by qualitative and relative quantitative analysis of PE species presented in liver samples from rats fed different diets. Using the ASID-DNLS-Shotgun ESI-MS/MS method, 45 PE species from rat livers have been identified and quantified in an efficient manner. The level of total PEs tended to decrease in the livers of rats on high fat diets compared with controls. The levels of PE 32:1, 34:3, 34:2, 36:3, 36:2, 42:10, plasmalogen PE 36:1 and lyso PE 22:6 were significantly reduced, while levels of PE 36:1 and lyso PE 16:0 increased. Copyright © 2015 Elsevier B.V. All rights reserved.

Heat-Treatment-Responsive Proteins in Different Developmental Stages of Tomato Pollen Detected by Targeted Mass Accuracy Precursor Alignment (tMAPA).

PubMed

Chaturvedi, Palak; Doerfler, Hannes; Jegadeesan, Sridharan; Ghatak, Arindam; Pressman, Etan; Castillejo, Maria Angeles; Wienkoop, Stefanie; Egelhofer, Volker; Firon, Nurit; Weckwerth, Wolfram

2015-11-06

Recently, we have developed a quantitative shotgun proteomics strategy called mass accuracy precursor alignment (MAPA). The MAPA algorithm uses high mass accuracy to bin mass-to-charge (m/z) ratios of precursor ions from LC-MS analyses, determines their intensities, and extracts a quantitative sample versus m/z ratio data alignment matrix from a multitude of samples. Here, we introduce a novel feature of this algorithm that allows the extraction and alignment of proteotypic peptide precursor ions or any other target peptide from complex shotgun proteomics data for accurate quantification of unique proteins. This strategy circumvents the problem of confusing the quantification of proteins due to indistinguishable protein isoforms by a typical shotgun proteomics approach. We applied this strategy to a comparison of control and heat-treated tomato pollen grains at two developmental stages, post-meiotic and mature. Pollen is a temperature-sensitive tissue involved in the reproductive cycle of plants and plays a major role in fruit setting and yield. By LC-MS-based shotgun proteomics, we identified more than 2000 proteins in total for all different tissues. By applying the targeted MAPA data-processing strategy, 51 unique proteins were identified as heat-treatment-responsive protein candidates. The potential function of the identified candidates in a specific developmental stage is discussed.

The first Taxus rhizosphere microbiome revealed by shotgun metagenomic sequencing.

PubMed

Hao, Da-Cheng; Zhang, Cai-Rong; Xiao, Pei-Gen

2018-06-01

In the present study, the shotgun high throughput metagenomic sequencing was implemented to globally capture the features of Taxus rhizosphere microbiome. Total reads could be assigned to 6925 species belonging to 113 bacteria phyla and 301 species of nine fungi phyla. For archaea and virus, 263 and 134 species were for the first time identified, respectively. More than 720,000 Unigenes were identified by clean reads assembly. The top five assigned phyla were Actinobacteria (363,941 Unigenes), Proteobacteria (182,053), Acidobacteria (44,527), Ascomycota (fungi; 18,267), and Chloroflexi (15,539). KEGG analysis predicted numerous functional genes; 7101 Unigenes belong to "Xenobiotics biodegradation and metabolism." A total of 12,040 Unigenes involved in defense mechanisms (e.g., xenobiotic metabolism) were annotated by eggNOG. Talaromyces addition could influence not only the diversity and structure of microbial communities of Taxus rhizosphere, but also the relative abundance of functional genes, including metabolic genes, antibiotic resistant genes, and genes involved in pathogen-host interaction, bacterial virulence, and bacterial secretion system. The structure and function of rhizosphere microbiome could be sensitive to non-native microbe addition, which could impact on the pollutant degradation. This study, complementary to the amplicon sequencing, more objectively reflects the native microbiome of Taxus rhizosphere and its response to environmental pressure, and lays a foundation for potential combination of phytoremediation and bioaugmentation. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An Integrated Proteomics/Transcriptomics Approach Points to Oxygen as the Main Electron Sink for Methanol Metabolism in Methylotenera mobilis▿†

PubMed Central

Beck, David A. C.; Hendrickson, Erik L.; Vorobev, Alexey; Wang, Tiansong; Lim, Sujung; Kalyuzhnaya, Marina G.; Lidstrom, Mary E.; Hackett, Murray; Chistoserdova, Ludmila

2011-01-01

Methylotenera species, unlike their close relatives in the genera Methylophilus, Methylobacillus, and Methylovorus, neither exhibit the activity of methanol dehydrogenase nor possess mxaFI genes encoding this enzyme, yet they are able to grow on methanol. In this work, we integrated a genome-wide proteomics approach, shotgun proteomics, and a genome-wide transcriptomics approach, shotgun transcriptome sequencing (RNA-seq), of Methylotenera mobilis JLW8 to identify genes and enzymes potentially involved in methanol oxidation, with special attention to alternative nitrogen sources, to address the question of whether nitrate could play a role as an electron acceptor in place of oxygen. Both proteomics and transcriptomics identified a limited number of genes and enzymes specifically responding to methanol. This set includes genes involved in oxidative stress response systems, a number of oxidoreductases, including XoxF-type alcohol dehydrogenases, a type II secretion system, and proteins without a predicted function. Nitrate stimulated expression of some genes in assimilatory nitrate reduction and denitrification pathways, while ammonium downregulated some of the nitrogen metabolism genes. However, none of these genes appeared to respond to methanol, which suggests that oxygen may be the main electron sink during growth on methanol. This study identifies initial targets for future focused physiological studies, including mutant analysis, which will provide further details into this novel process. PMID:21764938

Shotgun Proteomic Analysis of Plasma from Dairy Cattle Suffering from Footrot: Characterization of Potential Disease-Associated Factors

PubMed Central

Sun, Dongbo; Zhang, Hong; Guo, Donghua; Sun, Anguo; Wang, Hongbin

2013-01-01

The plasma proteome of healthy dairy cattle and those with footrot was investigated using a shotgun LC-MS/MS approach. In total, 648 proteins were identified in healthy plasma samples, of which 234 were non-redundant proteins and 123 were high-confidence proteins; 712 proteins were identified from footrot plasma samples, of which 272 were non-redundant proteins and 138 were high-confidence proteins. The high-confidence proteins showed significant differences between healthy and footrot plasma samples in molecular weight, isoelectric points and the Gene Ontology categories. 22 proteins were found that may differentiate between the two sets of plasma proteins, of which 16 potential differential expression (PDE) proteins from footrot plasma involved in immunoglobulins, innate immune recognition molecules, acute phase proteins, regulatory proteins, and cell adhesion and cytoskeletal proteins; 6 PDE proteins from healthy plasma involved in regulatory proteins, cytoskeletal proteins and coagulation factors. Of these PDE proteins, haptoglobin, SERPINA10 protein, afamin precursor, haptoglobin precursor, apolipoprotein D, predicted peptidoglycan recognition protein L (PGRP-L) and keratan sulfate proteoglycan (KS-PG) were suggested to be potential footrot-associated factors. The PDE proteins PGRP-L and KS-PG were highlighted as potential biomarkers of footrot in cattle. The resulting protein lists and potential differentially expressed proteins may provide valuable information to increase understanding of plasma protein profiles in cattle and to assist studies of footrot-associated factors. PMID:23418487

WHAM!: a web-based visualization suite for user-defined analysis of metagenomic shotgun sequencing data.

PubMed

Devlin, Joseph C; Battaglia, Thomas; Blaser, Martin J; Ruggles, Kelly V

2018-06-25

Exploration of large data sets, such as shotgun metagenomic sequence or expression data, by biomedical experts and medical professionals remains as a major bottleneck in the scientific discovery process. Although tools for this purpose exist for 16S ribosomal RNA sequencing analysis, there is a growing but still insufficient number of user-friendly interactive visualization workflows for easy data exploration and figure generation. The development of such platforms for this purpose is necessary to accelerate and streamline microbiome laboratory research. We developed the Workflow Hub for Automated Metagenomic Exploration (WHAM!) as a web-based interactive tool capable of user-directed data visualization and statistical analysis of annotated shotgun metagenomic and metatranscriptomic data sets. WHAM! includes exploratory and hypothesis-based gene and taxa search modules for visualizing differences in microbial taxa and gene family expression across experimental groups, and for creating publication quality figures without the need for command line interface or in-house bioinformatics. WHAM! is an interactive and customizable tool for downstream metagenomic and metatranscriptomic analysis providing a user-friendly interface allowing for easy data exploration by microbiome and ecological experts to facilitate discovery in multi-dimensional and large-scale data sets.

Protein identification and quantification from riverbank grape, Vitis riparia: Comparing SDS-PAGE and FASP-GPF techniques for shotgun proteomic analysis.

PubMed

George, Iniga S; Fennell, Anne Y; Haynes, Paul A

2015-09-01

Protein sample preparation optimisation is critical for establishing reproducible high throughput proteomic analysis. In this study, two different fractionation sample preparation techniques (in-gel digestion and in-solution digestion) for shotgun proteomics were used to quantitatively compare proteins identified in Vitis riparia leaf samples. The total number of proteins and peptides identified were compared between filter aided sample preparation (FASP) coupled with gas phase fractionation (GPF) and SDS-PAGE methods. There was a 24% increase in the total number of reproducibly identified proteins when FASP-GPF was used. FASP-GPF is more reproducible, less expensive and a better method than SDS-PAGE for shotgun proteomics of grapevine samples as it significantly increases protein identification across biological replicates. Total peptide and protein information from the two fractionation techniques is available in PRIDE with the identifier PXD001399 (http://proteomecentral.proteomexchange.org/dataset/PXD001399). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Data on xylem sap proteins from Mn- and Fe-deficient tomato plants obtained using shotgun proteomics.

PubMed

Ceballos-Laita, Laura; Gutierrez-Carbonell, Elain; Takahashi, Daisuke; Abadía, Anunciación; Uemura, Matsuo; Abadía, Javier; López-Millán, Ana Flor

2018-04-01

This article contains consolidated proteomic data obtained from xylem sap collected from tomato plants grown in Fe- and Mn-sufficient control, as well as Fe-deficient and Mn-deficient conditions. Data presented here cover proteins identified and quantified by shotgun proteomics and Progenesis LC-MS analyses: proteins identified with at least two peptides and showing changes statistically significant (ANOVA; p ≤ 0.05) and above a biologically relevant selected threshold (fold ≥ 2) between treatments are listed. The comparison between Fe-deficient, Mn-deficient and control xylem sap samples using a multivariate statistical data analysis (Principal Component Analysis, PCA) is also included. Data included in this article are discussed in depth in the research article entitled "Effects of Fe and Mn deficiencies on the protein profiles of tomato ( Solanum lycopersicum) xylem sap as revealed by shotgun analyses" [1]. This dataset is made available to support the cited study as well to extend analyses at a later stage.

76 FR 7824 - Procurement List; Proposed Additions

Federal Register 2010, 2011, 2012, 2013, 2014

2011-02-11

... NSNs: 8465-00-NIB-0211--Pouch, Four 3-round magazines, M26 12-guage shotgun MASS, Camouflage 8465-00-NIB-0212--Pouch, Four 5-round magazines, M26 12-guage shotgun MASS, Camouflage 8465-00-NIB-0213--Soft carrying case, Shotgun, 3-round magazine, M26 12-guage shotgun MASS, Camouflage 8465-00-NIB-0214--Soft...

Abdominal shotgun trauma: A case report

PubMed Central

Toutouzas, Konstantinos G; Larentzakis, Andreas; Drimousis, Panagiotis; Riga, Maria; Theodorou, Dimitrios; Katsaragakis, Stylianos

2008-01-01

Introduction One of the most lethal mechanisms of injury is shotgun wound and particularly the abdominal one. Case presentation We report a case of a 45 years old male suffering abdominal shotgun trauma, who survived his injuries. Conclusion The management of the abdominal shotgun wounds is mainly dependent on clinical examination and clinical judgment, while requires advanced surgical skills. PMID:18625076

Propionibacterium acnes: Disease-Causing Agent or Common Contaminant? Detection in Diverse Patient Samples by Next-Generation Sequencing

PubMed Central

Friis-Nielsen, Jens; Vinner, Lasse; Hansen, Thomas Arn; Richter, Stine Raith; Fridholm, Helena; Herrera, Jose Alejandro Romero; Lund, Ole; Brunak, Søren; Izarzugaza, Jose M. G.; Mourier, Tobias; Nielsen, Lars Peter

2016-01-01

Propionibacterium acnes is the most abundant bacterium on human skin, particularly in sebaceous areas. P. acnes is suggested to be an opportunistic pathogen involved in the development of diverse medical conditions but is also a proven contaminant of human clinical samples and surgical wounds. Its significance as a pathogen is consequently a matter of debate. In the present study, we investigated the presence of P. acnes DNA in 250 next-generation sequencing data sets generated from 180 samples of 20 different sample types, mostly of cancerous origin. The samples were subjected to either microbial enrichment, involving nuclease treatment to reduce the amount of host nucleic acids, or shotgun sequencing. We detected high proportions of P. acnes DNA in enriched samples, particularly skin tissue-derived and other tissue samples, with the levels being higher in enriched samples than in shotgun-sequenced samples. P. acnes reads were detected in most samples analyzed, though the proportions in most shotgun-sequenced samples were low. Our results show that P. acnes can be detected in practically all sample types when molecular methods, such as next-generation sequencing, are employed. The possibility of contamination from the patient or other sources, including laboratory reagents or environment, should therefore always be considered carefully when P. acnes is detected in clinical samples. We advocate that detection of P. acnes always be accompanied by experiments validating the association between this bacterium and any clinical condition. PMID:26818667

Shotgun protein sequencing: assembly of peptide tandem mass spectra from mixtures of modified proteins.

PubMed

Bandeira, Nuno; Clauser, Karl R; Pevzner, Pavel A

2007-07-01

Despite significant advances in the identification of known proteins, the analysis of unknown proteins by MS/MS still remains a challenging open problem. Although Klaus Biemann recognized the potential of MS/MS for sequencing of unknown proteins in the 1980s, low throughput Edman degradation followed by cloning still remains the main method to sequence unknown proteins. The automated interpretation of MS/MS spectra has been limited by a focus on individual spectra and has not capitalized on the information contained in spectra of overlapping peptides. Indeed the powerful shotgun DNA sequencing strategies have not been extended to automated protein sequencing. We demonstrate, for the first time, the feasibility of automated shotgun protein sequencing of protein mixtures by utilizing MS/MS spectra of overlapping and possibly modified peptides generated via multiple proteases of different specificities. We validate this approach by generating highly accurate de novo reconstructions of multiple regions of various proteins in western diamondback rattlesnake venom. We further argue that shotgun protein sequencing has the potential to overcome the limitations of current protein sequencing approaches and thus catalyze the otherwise impractical applications of proteomics methodologies in studies of unknown proteins.

Shotgun proteomic analysis of Bombyx mori brain: emphasis on regulation of behavior and development of the nervous system.

PubMed

Wang, Guo-Bao; Zheng, Qin; Shen, Yun-Wang; Wu, Xiao-Feng

2016-02-01

The insect brain plays crucial roles in the regulation of growth and development and in all types of behavior. We used sodium dodecyl sulfate polyacrylamide gel electrophoresis and high-performance liquid chromatography - electron spray ionization tandem mass spectrometry (ESI-MS/MS) shotgun to identify the proteome of the silkworm brain, to investigate its protein composition and to understand their biological functions. A total of 2210 proteins with molecular weights in the range of 5.64-1539.82 kDa and isoelectric points in the range of 3.78-12.55 were identified. These proteins were annotated according to Gene Ontology Annotation into the categories of molecular function, biological process and cellular component. We characterized two categories of proteins: one includes behavior-related proteins involved in the regulation of behaviors, such as locomotion, reproduction and learning; the other consists of proteins related to the development or function of the nervous system. The identified proteins were classified into 283 different pathways according to KEGG analysis, including the PI3K-Akt signaling pathway which plays a crucial role in mediating survival signals in a wide range of neuronal cell types. This extensive protein profile provides a basis for further understanding of the physiological functions in the silkworm brain. © 2014 Institute of Zoology, Chinese Academy of Sciences.

The effect of intermediate clothing targets on shotgun ballistics.

PubMed

Cail, Kenneth; Klatt, Edward

2013-12-01

The ballistic properties of shotgun shells are complex because of multiple projectiles fired simultaneously that interact and spread out to affect their energy relayed to a human target. Intermediate targets such as clothing can affect penetration into tissues. We studied the effect of common clothing fabrics as intermediate targets on penetration of shotgun shell pellets, using ordnance gelatin to simulate soft tissue and thin cowhide to simulate skin. A standard 12-gauge shotgun with modified choke was used with no. 8 shot ammunition. We found that protection afforded by fabrics to reduce penetration of shotgun pellets into tissues was greater at increasing distance from the muzzle beyond 40 yd (36.6 m). The thicker denim and cotton fabrics provided slightly greater protection than polyester. This study demonstrates that clothing modifies the potential wound patterns to victims of shotgun injuries.

Sequencing the Genome of the Heirloom Watermelon Cultivar Charleston Gray

USDA-ARS?s Scientific Manuscript database

The genome of the watermelon cultivar Charleston Gray, a major heirloom which has been used in breeding programs of many watermelon cultivars, was sequenced. Our strategy involved a hybrid approach using the Illumina and 454/Titanium next-generation sequencing technologies. For Illumina, shotgun g...

Biofilm-Growing Bacteria Involved in the Corrosion of Concrete Wastewater Pipes: Protocols for Comparative Metagenomic Analyses

EPA Science Inventory

Advances in high-throughput next-generation sequencing (NGS) technology for direct sequencing of environmental DNA (i.e. shotgun metagenomics) is transforming the field of microbiology. NGS technologies are now regularly being applied in comparative metagenomic studies, which pr...

Weapon Performance Testing and Analysis: The MODI-PAC Round, the Number 4 Lead-Shot Round, and the Flying Baton

DTIC Science & Technology

1976-01-01

items. The items tested were the MODI-PAC, a proprietary item of Reming)on Arms Company, a standard 12 - gauge round of No. 4 lead shot, and an...to refrain from testing this item. Therefore, the final selection of items for testing were (1) the MODI-PAC, (2) a standard 12 - gauge shotgun round of...The first item evaluated was the MODI-PAC5. The MOQ1-PAC which standsfor “modified impact “ is a 12 - gauge shotgun shell loaded with approximately 320

A combined meta-barcoding and shotgun metagenomic analysis of spontaneous wine fermentation.

PubMed

Sternes, Peter R; Lee, Danna; Kutyna, Dariusz R; Borneman, Anthony R

2017-07-01

Wine is a complex beverage, comprising hundreds of metabolites produced through the action of yeasts and bacteria in fermenting grape must. Commercially, there is now a growing trend away from using wine yeast (Saccharomyces) starter cultures, toward the historic practice of uninoculated or "wild" fermentation, where the yeasts and bacteria associated with the grapes and/or winery perform the fermentation. It is the varied metabolic contributions of these numerous non-Saccharomyces species that are thought to impart complexity and desirable taste and aroma attributes to wild ferments in comparison to their inoculated counterparts. To map the microflora of spontaneous fermentation, metagenomic techniques were employed to characterize and monitor the progression of fungal species in 5 different wild fermentations. Both amplicon-based ribosomal DNA internal transcribed spacer (ITS) phylotyping and shotgun metagenomics were used to assess community structure across different stages of fermentation. While providing a sensitive and highly accurate means of characterizing the wine microbiome, the shotgun metagenomic data also uncovered a significant overabundance bias in the ITS phylotyping abundance estimations for the common non-Saccharomyces wine yeast genus Metschnikowia. By identifying biases such as that observed for Metschnikowia, abundance measurements from future ITS phylotyping datasets can be corrected to provide more accurate species representation. Ultimately, as more shotgun metagenomic and single-strain de novo assemblies for key wine species become available, the accuracy of both ITS-amplicon and shotgun studies will greatly increase, providing a powerful methodology for deciphering the influence of the microbial community on the wine flavor and aroma. © The Authors 2017. Published by Oxford University Press.

A combined meta-barcoding and shotgun metagenomic analysis of spontaneous wine fermentation

PubMed Central

Sternes, Peter R.; Lee, Danna; Kutyna, Dariusz R.

2017-01-01

Abstract Wine is a complex beverage, comprising hundreds of metabolites produced through the action of yeasts and bacteria in fermenting grape must. Commercially, there is now a growing trend away from using wine yeast (Saccharomyces) starter cultures, toward the historic practice of uninoculated or “wild” fermentation, where the yeasts and bacteria associated with the grapes and/or winery perform the fermentation. It is the varied metabolic contributions of these numerous non-Saccharomyces species that are thought to impart complexity and desirable taste and aroma attributes to wild ferments in comparison to their inoculated counterparts. To map the microflora of spontaneous fermentation, metagenomic techniques were employed to characterize and monitor the progression of fungal species in 5 different wild fermentations. Both amplicon-based ribosomal DNA internal transcribed spacer (ITS) phylotyping and shotgun metagenomics were used to assess community structure across different stages of fermentation. While providing a sensitive and highly accurate means of characterizing the wine microbiome, the shotgun metagenomic data also uncovered a significant overabundance bias in the ITS phylotyping abundance estimations for the common non-Saccharomyces wine yeast genus Metschnikowia. By identifying biases such as that observed for Metschnikowia, abundance measurements from future ITS phylotyping datasets can be corrected to provide more accurate species representation. Ultimately, as more shotgun metagenomic and single-strain de novo assemblies for key wine species become available, the accuracy of both ITS-amplicon and shotgun studies will greatly increase, providing a powerful methodology for deciphering the influence of the microbial community on the wine flavor and aroma. PMID:28595314

Studies of a biochemical factory: tomato trichome deep expressed sequence tag sequencing and proteomics.

PubMed

Schilmiller, Anthony L; Miner, Dennis P; Larson, Matthew; McDowell, Eric; Gang, David R; Wilkerson, Curtis; Last, Robert L

2010-07-01

Shotgun proteomics analysis allows hundreds of proteins to be identified and quantified from a single sample at relatively low cost. Extensive DNA sequence information is a prerequisite for shotgun proteomics, and it is ideal to have sequence for the organism being studied rather than from related species or accessions. While this requirement has limited the set of organisms that are candidates for this approach, next generation sequencing technologies make it feasible to obtain deep DNA sequence coverage from any organism. As part of our studies of specialized (secondary) metabolism in tomato (Solanum lycopersicum) trichomes, 454 sequencing of cDNA was combined with shotgun proteomics analyses to obtain in-depth profiles of genes and proteins expressed in leaf and stem glandular trichomes of 3-week-old plants. The expressed sequence tag and proteomics data sets combined with metabolite analysis led to the discovery and characterization of a sesquiterpene synthase that produces beta-caryophyllene and alpha-humulene from E,E-farnesyl diphosphate in trichomes of leaf but not of stem. This analysis demonstrates the utility of combining high-throughput cDNA sequencing with proteomics experiments in a target tissue. These data can be used for dissection of other biochemical processes in these specialized epidermal cells.

Studies of a Biochemical Factory: Tomato Trichome Deep Expressed Sequence Tag Sequencing and Proteomics1[W][OA

PubMed Central

Schilmiller, Anthony L.; Miner, Dennis P.; Larson, Matthew; McDowell, Eric; Gang, David R.; Wilkerson, Curtis; Last, Robert L.

2010-01-01

Shotgun proteomics analysis allows hundreds of proteins to be identified and quantified from a single sample at relatively low cost. Extensive DNA sequence information is a prerequisite for shotgun proteomics, and it is ideal to have sequence for the organism being studied rather than from related species or accessions. While this requirement has limited the set of organisms that are candidates for this approach, next generation sequencing technologies make it feasible to obtain deep DNA sequence coverage from any organism. As part of our studies of specialized (secondary) metabolism in tomato (Solanum lycopersicum) trichomes, 454 sequencing of cDNA was combined with shotgun proteomics analyses to obtain in-depth profiles of genes and proteins expressed in leaf and stem glandular trichomes of 3-week-old plants. The expressed sequence tag and proteomics data sets combined with metabolite analysis led to the discovery and characterization of a sesquiterpene synthase that produces β-caryophyllene and α-humulene from E,E-farnesyl diphosphate in trichomes of leaf but not of stem. This analysis demonstrates the utility of combining high-throughput cDNA sequencing with proteomics experiments in a target tissue. These data can be used for dissection of other biochemical processes in these specialized epidermal cells. PMID:20431087

Midpregnancy Marriage and Divorce: Why the Death of Shotgun Marriage Has Been Greatly Exaggerated

PubMed Central

Ananat, Elizabeth O.; Gassman-Pines, Anna

2018-01-01

Conventional wisdom holds that births following the colloquially termed “shotgun marriage”—that is, births to parents who married between conception and the birth—are nearing obsolescence. To investigate trends in shotgun marriage, we matched North Carolina administrative data on nearly 800,000 first births among white and black mothers to marriage and divorce records. We found that among married births, midpregnancy-married births (our preferred term for shotgun-married births) have been relatively stable at about 10 % over the past quarter-century while increasing substantially for vulnerable population subgroups. In 2012, among black and white less-educated and younger women, midpregnancy-married births accounted for approximately 20 % to 25 % of married first births. The increasing representation of midpregnancy-married births among married births raises concerns about well-being among at-risk families because midpregnancy marriages may be quite fragile. Our analysis revealed, however, that midpregnancy marriages were more likely to dissolve only among more advantaged groups. Of those groups considered to be most at risk of divorce—namely, black women with lower levels of education and who were younger—midpregnancy marriages had the same or lower likelihood of divorce as preconception marriages. Our results suggest an overlooked resiliency in a type of marriage that has only increased in salience. PMID:27804061

Midpregnancy Marriage and Divorce: Why the Death of Shotgun Marriage Has Been Greatly Exaggerated.

PubMed

Gibson-Davis, Christina M; Ananat, Elizabeth O; Gassman-Pines, Anna

2016-12-01

Conventional wisdom holds that births following the colloquially termed "shotgun marriage"-that is, births to parents who married between conception and the birth-are nearing obsolescence. To investigate trends in shotgun marriage, we matched North Carolina administrative data on nearly 800,000 first births among white and black mothers to marriage and divorce records. We found that among married births, midpregnancy-married births (our preferred term for shotgun-married births) have been relatively stable at about 10 % over the past quarter-century while increasing substantially for vulnerable population subgroups. In 2012, among black and white less-educated and younger women, midpregnancy-married births accounted for approximately 20 % to 25 % of married first births. The increasing representation of midpregnancy-married births among married births raises concerns about well-being among at-risk families because midpregnancy marriages may be quite fragile. Our analysis revealed, however, that midpregnancy marriages were more likely to dissolve only among more advantaged groups. Of those groups considered to be most at risk of divorce-namely, black women with lower levels of education and who were younger-midpregnancy marriages had the same or lower likelihood of divorce as preconception marriages. Our results suggest an overlooked resiliency in a type of marriage that has only increased in salience.

Enhanced Lipidome Coverage in Shotgun Analyses by using Gas-Phase Fractionation

NASA Astrophysics Data System (ADS)

Nazari, Milad; Muddiman, David C.

2016-11-01

A high resolving power shotgun lipidomics strategy using gas-phase fractionation and data-dependent acquisition (DDA) was applied toward comprehensive characterization of lipids in a hen ovarian tissue in an untargeted fashion. Using this approach, a total of 822 unique lipids across a diverse range of lipid categories and classes were identified based on their MS/MS fragmentation patterns. Classes of glycerophospholipids and glycerolipids, such as glycerophosphocholines (PC), glycerophosphoethanolamines (PE), and triglycerides (TG), are often the most abundant peaks observed in shotgun lipidomics analyses. These ions suppress the signal from low abundance ions and hinder the chances of characterizing low abundant lipids when DDA is used. These issues were circumvented by utilizing gas-phase fractionation, where DDA was performed on narrow m/z ranges instead of a broad m/z range. Employing gas-phase fractionation resulted in an increase in sensitivity by more than an order of magnitude in both positive- and negative-ion modes. Furthermore, the enhanced sensitivity increased the number of lipids identified by a factor of ≈4, and facilitated identification of low abundant lipids from classes such as cardiolipins that are often difficult to observe in untargeted shotgun analyses and require sample-specific preparation steps prior to analysis. This method serves as a resource for comprehensive profiling of lipids from many different categories and classes in an untargeted manner, as well as for targeted and quantitative analyses of individual lipids. Furthermore, this comprehensive analysis of the lipidome can serve as a species- and tissue-specific database for confident identification of other MS-based datasets, such as mass spectrometry imaging.

Liver protein profiles in insulin receptor-knockout mice reveal novel molecules involved in the diabetes pathophysiology.

PubMed

Capuani, Barbara; Della-Morte, David; Donadel, Giulia; Caratelli, Sara; Bova, Luca; Pastore, Donatella; De Canio, Michele; D'Aguanno, Simona; Coppola, Andrea; Pacifici, Francesca; Arriga, Roberto; Bellia, Alfonso; Ferrelli, Francesca; Tesauro, Manfredi; Federici, Massimo; Neri, Anna; Bernardini, Sergio; Sbraccia, Paolo; Di Daniele, Nicola; Sconocchia, Giuseppe; Orlandi, Augusto; Urbani, Andrea; Lauro, Davide

2015-05-01

Liver has a principal role in glucose regulation and lipids homeostasis. It is under a complex control by substrates such as hormones, nutrients, and neuronal impulses. Insulin promotes glycogen synthesis, lipogenesis, and lipoprotein synthesis and inhibits gluconeogenesis, glycogenolysis, and VLDL secretion by modifying the expression and enzymatic activity of specific molecules. To understand the pathophysiological mechanisms leading to metabolic liver disease, we analyzed liver protein patterns expressed in a mouse model of diabetes by proteomic approaches. We used insulin receptor-knockout (IR(-/-)) and heterozygous (IR(+/-)) mice as a murine model of liver metabolic dysfunction associated with diabetic ketoacidosis and insulin resistance. We evaluated liver fatty acid levels by microscopic examination and protein expression profiles by orthogonal experimental strategies using protein 2-DE MALDI-TOF/TOF and peptic nLC-MS/MS shotgun profiling. Identified proteins were then loaded into Ingenuity Pathways Analysis to find possible molecular networks. Twenty-eight proteins identified by 2-DE analysis and 24 identified by nLC-MS/MS shotgun were differentially expressed among the three genotypes. Bioinformatic analysis revealed a central role of high-mobility group box 1/2 and huntigtin never reported before in association with metabolic and related liver disease. A different modulation of these proteins in both blood and hepatic tissue further suggests their role in these processes. These results provide new insight into pathophysiology of insulin resistance and hepatic steatosis and could be useful in identifying novel biomarkers to predict risk for diabetes and its complications. Copyright © 2015 the American Physiological Society.

THE OPPORTUNISTIC PATHOGEN TOXOPLASMA GONDII DEPLOYS A DIVERSE LEGION OF INVASION AND SURVIVAL PROTEINS

PubMed Central

Zhou, Xing W.; Kafsack, Björn F. C.; Cole, Robert N.; Beckett, Phil; Shen, Rong F.; Carruthers, Vern B.

2006-01-01

Host cell invasion is an essential step during infection by Toxoplasma gondii, an intracellular protozoan that causes the severe opportunistic disease toxoplasmosis in humans. Recent evidence strongly suggests that proteins discharged from Toxoplasma apical secretory organelles (micronemes, dense granules, and rhoptries) play key roles in host cell invasion and survival during infection. However, to date, only a limited number of secretory proteins have been discovered and the full spectrum of effector molecules involved in parasite invasion and survival remains unknown. To address these issues, we analyzed a large cohort of freely released Toxoplasma secretory proteins using two complementary methodologies, 2-DE/MS and LC/ESI-MS-MS (MudPIT, shotgun proteomics). Visualization of Toxoplasma secretory products by 2-DE revealed ∼100 spots, most of which were successfully identified by protein microsequencing or MALDI-MS analysis. Many proteins were present in multiple species suggesting they are subjected to substantial posttranslational modification. Shotgun proteomic analysis of the secretory fraction revealed several additional products including novel putative adhesive proteins, proteases, and hypothetical secretory proteins similar to products expressed by other related parasites including Plasmodium, the etiologic agent of malaria. A subset of novel proteins were re-expressed as fusions to yellow fluorescent protein and this initial screen revealed shared and distinct localizations within secretory compartments of T. gondii tachyzoites. The findings provide a uniquely broad view of Toxoplasma secretory proteins that participate in parasite survival and pathogenesis during infection. PMID:16002397

The genome of flax (Linum usitatissimum) assembled de novo from short shotgun sequence reads.

PubMed

Wang, Zhiwen; Hobson, Neil; Galindo, Leonardo; Zhu, Shilin; Shi, Daihu; McDill, Joshua; Yang, Linfeng; Hawkins, Simon; Neutelings, Godfrey; Datla, Raju; Lambert, Georgina; Galbraith, David W; Grassa, Christopher J; Geraldes, Armando; Cronk, Quentin C; Cullis, Christopher; Dash, Prasanta K; Kumar, Polumetla A; Cloutier, Sylvie; Sharpe, Andrew G; Wong, Gane K-S; Wang, Jun; Deyholos, Michael K

2012-11-01

Flax (Linum usitatissimum) is an ancient crop that is widely cultivated as a source of fiber, oil and medicinally relevant compounds. To accelerate crop improvement, we performed whole-genome shotgun sequencing of the nuclear genome of flax. Seven paired-end libraries ranging in size from 300 bp to 10 kb were sequenced using an Illumina genome analyzer. A de novo assembly, comprised exclusively of deep-coverage (approximately 94× raw, approximately 69× filtered) short-sequence reads (44-100 bp), produced a set of scaffolds with N(50) =694 kb, including contigs with N(50)=20.1 kb. The contig assembly contained 302 Mb of non-redundant sequence representing an estimated 81% genome coverage. Up to 96% of published flax ESTs aligned to the whole-genome shotgun scaffolds. However, comparisons with independently sequenced BACs and fosmids showed some mis-assembly of regions at the genome scale. A total of 43384 protein-coding genes were predicted in the whole-genome shotgun assembly, and up to 93% of published flax ESTs, and 86% of A. thaliana genes aligned to these predicted genes, indicating excellent coverage and accuracy at the gene level. Analysis of the synonymous substitution rates (K(s) ) observed within duplicate gene pairs was consistent with a recent (5-9 MYA) whole-genome duplication in flax. Within the predicted proteome, we observed enrichment of many conserved domains (Pfam-A) that may contribute to the unique properties of this crop, including agglutinin proteins. Together these results show that de novo assembly, based solely on whole-genome shotgun short-sequence reads, is an efficient means of obtaining nearly complete genome sequence information for some plant species. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

PatternLab for proteomics 4.0: A one-stop shop for analyzing shotgun proteomic data

PubMed Central

Carvalho, Paulo C; Lima, Diogo B; Leprevost, Felipe V; Santos, Marlon D M; Fischer, Juliana S G; Aquino, Priscila F; Moresco, James J; Yates, John R; Barbosa, Valmir C

2017-01-01

PatternLab for proteomics is an integrated computational environment that unifies several previously published modules for analyzing shotgun proteomic data. PatternLab contains modules for formatting sequence databases, performing peptide spectrum matching, statistically filtering and organizing shotgun proteomic data, extracting quantitative information from label-free and chemically labeled data, performing statistics for differential proteomics, displaying results in a variety of graphical formats, performing similarity-driven studies with de novo sequencing data, analyzing time-course experiments, and helping with the understanding of the biological significance of data in the light of the Gene Ontology. Here we describe PatternLab for proteomics 4.0, which closely knits together all of these modules in a self-contained environment, covering the principal aspects of proteomic data analysis as a freely available and easily installable software package. All updates to PatternLab, as well as all new features added to it, have been tested over the years on millions of mass spectra. PMID:26658470

27 CFR 478.39 - Assembly of semiautomatic rifles or shotguns.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 27 Alcohol, Tobacco Products and Firearms 3 2011-04-01 2010-04-01 true Assembly of semiautomatic... AMMUNITION Administrative and Miscellaneous Provisions § 478.39 Assembly of semiautomatic rifles or shotguns.... (b) The provisions of this section shall not apply to: (1) The assembly of such rifle or shotgun for...

27 CFR 478.39 - Assembly of semiautomatic rifles or shotguns.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 27 Alcohol, Tobacco Products and Firearms 3 2014-04-01 2014-04-01 false Assembly of semiautomatic... AMMUNITION Administrative and Miscellaneous Provisions § 478.39 Assembly of semiautomatic rifles or shotguns.... (b) The provisions of this section shall not apply to: (1) The assembly of such rifle or shotgun for...

27 CFR 478.39 - Assembly of semiautomatic rifles or shotguns.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 3 2010-04-01 2010-04-01 false Assembly of semiautomatic... AMMUNITION Administrative and Miscellaneous Provisions § 478.39 Assembly of semiautomatic rifles or shotguns.... (b) The provisions of this section shall not apply to: (1) The assembly of such rifle or shotgun for...

27 CFR 478.39 - Assembly of semiautomatic rifles or shotguns.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 27 Alcohol, Tobacco Products and Firearms 3 2013-04-01 2013-04-01 false Assembly of semiautomatic... AMMUNITION Administrative and Miscellaneous Provisions § 478.39 Assembly of semiautomatic rifles or shotguns.... (b) The provisions of this section shall not apply to: (1) The assembly of such rifle or shotgun for...

27 CFR 478.39 - Assembly of semiautomatic rifles or shotguns.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 27 Alcohol, Tobacco Products and Firearms 3 2012-04-01 2010-04-01 true Assembly of semiautomatic... AMMUNITION Administrative and Miscellaneous Provisions § 478.39 Assembly of semiautomatic rifles or shotguns.... (b) The provisions of this section shall not apply to: (1) The assembly of such rifle or shotgun for...

Look away: arterial and venous intravascular embolisation following shotgun injury.

PubMed

Vedelago, John; Dick, Elizabeth; Thomas, Robert; Jones, Brynmor; Kirmi, Olga; Becker, Jennifer; Alavi, Afshin; Gedroyc, Wladyslaw

2014-01-01

We describe two cases of intravascular embolization of shotgun pellets found distant to the entry site of penetrating firearm injury. The cases demonstrate antegrade embolization of a shotgun pellet from neck to right middle cerebral artery, and antegrade followed by retrograde venous embolization through the left lower limb to pelvis. Radiologists and Trauma Physicians should be aware that post shotgun injury, the likelihood of an embolised shot pellet is increased compared to other types of firearm missile injury, and should therefore search away from the site of injury to find such missiles. Shotgun pellets may travel in an antegrade or a retrograde intravascular direction - both were seen in these cases - and may not be clinically obvious. This underscores the importance of a meticuluous search through all images, including CT scout images, for evidence of their presence.

Reser­voir anisotropy and facies stratigraphic framework in the Pale­ocene Fort Union Formation, western Wind River basin

USGS Publications Warehouse

Flores, R.M.; Keighin, C.W.

1993-01-01

Investigation of reservoir anisotropy and lithofacies stratigraphic framework in the Fort Union Formation in western Wind River Basin, Wyoming focused on excellent surface exposures in the Shotgun Butte, Eagle Point, and Shotgun Bench synclines, and in the Merriam anticline area of the Wind River Reservation (Fig. 1). A complementary study was made of the formation in the Muddy Ridge and Pavillion gas fields, 8-10 mi to the southeast (Fig. 2). The Fort Union Formation is as much as 4000 ft thick in these areas, but thins to approximately 1800 ft toward the northern flank of the Little Dome anticline 3 mi south of Merriam anticline (Keefer and Troyer, 1964). The Fort Union Formation includes interbedded conglomerates, sandstones, siltstones, mudstones, coals, and carbonaceous shales (Fig. 3). The lower member of the Fort Union Formation is dominated by conglomerates and sandstones. The overlying Shotgun Member of the Fort Union Formation mainly consists of siltstones, mudstones, and carbonaceous shales, and coals, and subordinate sandstones. Contact between the lower member and Shotgun Member is gradational and marked by a topographic change from the resistant conglomerates and sandstones of the lower member to less resistant fine-grained strata of the Shotgun Member. In addition, the Shotgun Member commonly contains coal and carbonaceous shale beds, both in the surface and subsurface (Fig. 4). About 15-20 mi east of the study area the Waltman Shale Member of the Fort Union Formation pinches out at the contact between the lower member and Shotgun Member (Keefer and Johnson, this volume). The Waltman Shale Member, which consists of brown to gray silty and shaly claystones interbedded with sandstones, increases in thickness to as much as 3000 ft eastward into the basin center (Keefer, 1961; 1965). Thus, eastward, the Paleocene Fort Union Formation in ascending order, contains the lower member, Waltman Shale Member, and Shotgun Member. The Shotgun Member generally thins and interfingers with the Waltman Member.

Entrance, exit, and reentrance of one shot with a shotgun.

PubMed

Gulmann, C; Hougen, H P

1999-03-01

The case being reported is one of a homicidal shotgun fatality with an unusual wound pattern. A 34-year-old man was shot at close range with a 12-gauge shotgun armed with No. 5 birdshot ammunition. The shot entered the left axillary region, exited through the left infraclavicular region, and thereafter penetrated the left side of the neck, causing tearing of the left common carotid artery and the right internal carotid artery. The entrance wound in the axilla was larger than the other wounds, and before autopsy it was believed that the shotgun had been fired twice, causing one wound in the neck and one wound perforating the infraclavicular region and exiting through the left axillary region. Thus, this case shows that unusual wound patterns in shotgun fatalities can easily lead to incorrect assumptions with regard to number and direction of shots fired unless thorough investigation is carried out postmortem.

Characterization of a Protein Interactome by Co-Immunoprecipitation and Shotgun Mass Spectrometry.

PubMed

Maccarrone, Giuseppina; Bonfiglio, Juan Jose; Silberstein, Susana; Turck, Christoph W; Martins-de-Souza, Daniel

2017-01-01

Identifying the partners of a given protein (the interactome) may provide leads about the protein's function and the molecular mechanisms in which it is involved. One of the alternative strategies used to characterize protein interactomes consists of co-immunoprecipitation (co-IP) followed by shotgun mass spectrometry. This enables the isolation and identification of a protein target in its native state and its interactome from cells or tissue lysates under physiological conditions. In this chapter, we describe a co-IP protocol for interactome studies that uses an antibody against a protein of interest bound to protein A/G plus agarose beads to isolate a protein complex. The interacting proteins may be further fractionated by SDS-PAGE, followed by in-gel tryptic digestion and nano liquid chromatography high-resolution tandem mass spectrometry (nLC ESI-MS/MS) for identification purposes. The computational tools, strategy for protein identification, and use of interactome databases also will be described.

Evaluation of "shotgun" proteomics for identification of biological threat agents in complex environmental matrixes: experimental simulations.

PubMed

Verberkmoes, Nathan C; Hervey, W Judson; Shah, Manesh; Land, Miriam; Hauser, Loren; Larimer, Frank W; Van Berkel, Gary J; Goeringer, Douglas E

2005-02-01

There is currently a great need for rapid detection and positive identification of biological threat agents, as well as microbial species in general, directly from complex environmental samples. This need is most urgent in the area of homeland security, but also extends into medical, environmental, and agricultural sciences. Mass-spectrometry-based analysis is one of the leading technologies in the field with a diversity of different methodologies for biothreat detection. Over the past few years, "shotgun"proteomics has become one method of choice for the rapid analysis of complex protein mixtures by mass spectrometry. Recently, it was demonstrated that this methodology is capable of distinguishing a target species against a large database of background species from a single-component sample or dual-component mixtures with relatively the same concentration. Here, we examine the potential of shotgun proteomics to analyze a target species in a background of four contaminant species. We tested the capability of a common commercial mass-spectrometry-based shotgun proteomics platform for the detection of the target species (Escherichia coli) at four different concentrations and four different time points of analysis. We also tested the effect of database size on positive identification of the four microbes used in this study by testing a small (13-species) database and a large (261-species) database. The results clearly indicated that this technology could easily identify the target species at 20% in the background mixture at a 60, 120, 180, or 240 min analysis time with the small database. The results also indicated that the target species could easily be identified at 20% or 6% but could not be identified at 0.6% or 0.06% in either a 240 min analysis or a 30 h analysis with the small database. The effects of the large database were severe on the target species where detection above the background at any concentration used in this study was impossible, though the three other microbes used in this study were clearly identified above the background when analyzed with the large database. This study points to the potential application of this technology for biological threat agent detection but highlights many areas of needed research before the technology will be useful in real world samples.

Computational approaches to protein inference in shotgun proteomics

PubMed Central

2012-01-01

Shotgun proteomics has recently emerged as a powerful approach to characterizing proteomes in biological samples. Its overall objective is to identify the form and quantity of each protein in a high-throughput manner by coupling liquid chromatography with tandem mass spectrometry. As a consequence of its high throughput nature, shotgun proteomics faces challenges with respect to the analysis and interpretation of experimental data. Among such challenges, the identification of proteins present in a sample has been recognized as an important computational task. This task generally consists of (1) assigning experimental tandem mass spectra to peptides derived from a protein database, and (2) mapping assigned peptides to proteins and quantifying the confidence of identified proteins. Protein identification is fundamentally a statistical inference problem with a number of methods proposed to address its challenges. In this review we categorize current approaches into rule-based, combinatorial optimization and probabilistic inference techniques, and present them using integer programing and Bayesian inference frameworks. We also discuss the main challenges of protein identification and propose potential solutions with the goal of spurring innovative research in this area. PMID:23176300

Analysis of sequence variability in the macronuclear DNA of Paramecium tetraurelia: A somatic view of the germline

PubMed Central

Duret, Laurent; Cohen, Jean; Jubin, Claire; Dessen, Philippe; Goût, Jean-François; Mousset, Sylvain; Aury, Jean-Marc; Jaillon, Olivier; Noël, Benjamin; Arnaiz, Olivier; Bétermier, Mireille; Wincker, Patrick; Meyer, Eric; Sperling, Linda

2008-01-01

Ciliates are the only unicellular eukaryotes known to separate germinal and somatic functions. Diploid but silent micronuclei transmit the genetic information to the next sexual generation. Polyploid macronuclei express the genetic information from a streamlined version of the genome but are replaced at each sexual generation. The macronuclear genome of Paramecium tetraurelia was recently sequenced by a shotgun approach, providing access to the gene repertoire. The 72-Mb assembly represents a consensus sequence for the somatic DNA, which is produced after sexual events by reproducible rearrangements of the zygotic genome involving elimination of repeated sequences, precise excision of unique-copy internal eliminated sequences (IES), and amplification of the cellular genes to high copy number. We report use of the shotgun sequencing data (>106 reads representing 13× coverage of a completely homozygous clone) to evaluate variability in the somatic DNA produced by these developmental genome rearrangements. Although DNA amplification appears uniform, both of the DNA elimination processes produce sequence heterogeneity. The variability that arises from IES excision allowed identification of hundreds of putative new IESs, compared to 42 that were previously known, and revealed cases of erroneous excision of segments of coding sequences. We demonstrate that IESs in coding regions are under selective pressure to introduce premature termination of translation in case of excision failure. PMID:18256234

A Shotgun Proteomic Approach Reveals That Fe Deficiency Causes Marked Changes in the Protein Profiles of Plasma Membrane and Detergent-Resistant Microdomain Preparations from Beta vulgaris Roots.

PubMed

Gutierrez-Carbonell, Elain; Takahashi, Daisuke; Lüthje, Sabine; González-Reyes, José Antonio; Mongrand, Sébastien; Contreras-Moreira, Bruno; Abadía, Anunciación; Uemura, Matsuo; Abadía, Javier; López-Millán, Ana Flor

2016-08-05

In the present study we have used label-free shotgun proteomic analysis to examine the effects of Fe deficiency on the protein profiles of highly pure sugar beet root plasma membrane (PM) preparations and detergent-resistant membranes (DRMs), the latter as an approach to study microdomains. Altogether, 545 proteins were detected, with 52 and 68 of them changing significantly with Fe deficiency in PM and DRM, respectively. Functional categorization of these proteins showed that signaling and general and vesicle-related transport accounted for approximately 50% of the differences in both PM and DRM, indicating that from a qualitative point of view changes induced by Fe deficiency are similar in both preparations. Results indicate that Fe deficiency has an impact in phosphorylation processes at the PM level and highlight the involvement of signaling proteins, especially those from the 14-3-3 family. Lipid profiling revealed Fe-deficiency-induced decreases in phosphatidic acid derivatives, which may impair vesicle formation, in agreement with the decreases measured in proteins related to intracellular trafficking and secretion. The modifications induced by Fe deficiency in the relative enrichment of proteins in DRMs revealed the existence of a group of cytoplasmic proteins that appears to be more attached to the PM in conditions of Fe deficiency.

Insights into microbial involvement in desert varnish formation retrieved from metagenomic analysis.

PubMed

Lang-Yona, Naama; Maier, Stefanie; Macholdt, Dorothea S; Müller-Germann, Isabell; Yordanova, Petya; Rodriguez-Caballero, Emilio; Jochum, Klaus P; Al-Amri, Abdullah; Andreae, Meinrat O; Fröhlich-Nowoisky, Janine; Weber, Bettina

2018-02-28

Desert varnishes are dark rock coatings observed in arid environments and might resemble Mn-rich coatings found on Martian rocks. Their formation mechanism is not fully understood and the possible microbial involvement is under debate. In this study, we applied DNA metagenomic Shotgun sequencing of varnish and surrounding soil to evaluate the composition of the microbial community and its potential metabolic function. We found that the α diversity was lower in varnish compared to soil samples (p value < 0.05), suggesting distinct populations with significantly higher abundance of Actinobacteria, Proteobacteria and Cyanobacteria within the varnish. Additionally, we observed increased levels of transition metal metabolic processes in varnish compared to soil samples. Nevertheless, potentially relevant enzymes for varnish formation were detected at low to insignificant levels in both niches, indicating no current direct microbial involvement in Mn oxidation. This finding is supported by quantitative genomic analysis, elemental analysis, fluorescence imaging and scanning transmission X-ray microscopy. We thus conclude that the distinct microbial communities detected in desert varnish originate from settled Aeolian microbes, which colonized this nutrient-enriched niche, and discuss possible indirect contributions of microorganisms to the formation of desert varnish. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

Comparison of normalization methods for the analysis of metagenomic gene abundance data.

PubMed

Pereira, Mariana Buongermino; Wallroth, Mikael; Jonsson, Viktor; Kristiansson, Erik

2018-04-20

In shotgun metagenomics, microbial communities are studied through direct sequencing of DNA without any prior cultivation. By comparing gene abundances estimated from the generated sequencing reads, functional differences between the communities can be identified. However, gene abundance data is affected by high levels of systematic variability, which can greatly reduce the statistical power and introduce false positives. Normalization, which is the process where systematic variability is identified and removed, is therefore a vital part of the data analysis. A wide range of normalization methods for high-dimensional count data has been proposed but their performance on the analysis of shotgun metagenomic data has not been evaluated. Here, we present a systematic evaluation of nine normalization methods for gene abundance data. The methods were evaluated through resampling of three comprehensive datasets, creating a realistic setting that preserved the unique characteristics of metagenomic data. Performance was measured in terms of the methods ability to identify differentially abundant genes (DAGs), correctly calculate unbiased p-values and control the false discovery rate (FDR). Our results showed that the choice of normalization method has a large impact on the end results. When the DAGs were asymmetrically present between the experimental conditions, many normalization methods had a reduced true positive rate (TPR) and a high false positive rate (FPR). The methods trimmed mean of M-values (TMM) and relative log expression (RLE) had the overall highest performance and are therefore recommended for the analysis of gene abundance data. For larger sample sizes, CSS also showed satisfactory performance. This study emphasizes the importance of selecting a suitable normalization methods in the analysis of data from shotgun metagenomics. Our results also demonstrate that improper methods may result in unacceptably high levels of false positives, which in turn may lead to incorrect or obfuscated biological interpretation.

Boron nitride as desalting material in combination with phosphopeptide enrichment in shotgun proteomics.

PubMed

Furuhashi, Takeshi; Nukarinen, Ella; Ota, Shigenori; Weckwerth, Wolfram

2014-05-01

Hydrophilic peptides in shotgun proteomics have been shown to be problematic in conventional chromatography. Typically, C18 solid phase extraction or peptide traps are used for desalting the sample prior to mass spectrometry analysis, but the capacity to retain hydrophilic peptides is not very high, causing a bias toward more hydrophobic peptides. This is particularly problematic in phosphoproteomic studies. We tested the compatibility of commercially available boron nitride as a novel material for peptide desalting. Boron nitride can be used to recover a wide range of peptides with different physicochemical properties comparable to combined C18 and graphite carbon material. Copyright © 2014. Published by Elsevier Inc.

Application of the whole-transcriptome shotgun sequencing approach to the study of Philadelphia-positive acute lymphoblastic leukemia

PubMed Central

Iacobucci, I; Ferrarini, A; Sazzini, M; Giacomelli, E; Lonetti, A; Xumerle, L; Ferrari, A; Papayannidis, C; Malerba, G; Luiselli, D; Boattini, A; Garagnani, P; Vitale, A; Soverini, S; Pane, F; Baccarani, M; Delledonne, M; Martinelli, G

2012-01-01

Although the pathogenesis of BCR–ABL1-positive acute lymphoblastic leukemia (ALL) is mainly related to the expression of the BCR–ABL1 fusion transcript, additional cooperating genetic lesions are supposed to be involved in its development and progression. Therefore, in an attempt to investigate the complex landscape of mutations, changes in expression profiles and alternative splicing (AS) events that can be observed in such disease, the leukemia transcriptome of a BCR–ABL1-positive ALL patient at diagnosis and at relapse was sequenced using a whole-transcriptome shotgun sequencing (RNA-Seq) approach. A total of 13.9 and 15.8 million sequence reads was generated from de novo and relapsed samples, respectively, and aligned to the human genome reference sequence. This led to the identification of five validated missense mutations in genes involved in metabolic processes (DPEP1, TMEM46), transport (MVP), cell cycle regulation (ABL1) and catalytic activity (CTSZ), two of which resulted in acquired relapse variants. In all, 6390 and 4671 putative AS events were also detected, as well as expression levels for 18 315 and 18 795 genes, 28% of which were differentially expressed in the two disease phases. These data demonstrate that RNA-Seq is a suitable approach for identifying a wide spectrum of genetic alterations potentially involved in ALL. PMID:22829256

Secondary intracranial subarachnoid hemorrhage due to spinal missile injury.

PubMed

Smialek, J E; Chason, J L; Kshirsagar, V; Spitz, W U

1981-04-01

Fresh intracranial subarachnoid hemorrhage may occur secondary to blast-type injury of the spinal cord. This phenomenon is demonstrated in four cases of gunshot and shotgun wounds involving the spinal column. The significance of such a finding is that the subarachnoid hemorrhage should not be construed to represent an independent injury. Such an erroneous conclusion could jeopardize a theory of self-defense in a homicidal shooting.

PAnalyzer: a software tool for protein inference in shotgun proteomics.

PubMed

Prieto, Gorka; Aloria, Kerman; Osinalde, Nerea; Fullaondo, Asier; Arizmendi, Jesus M; Matthiesen, Rune

2012-11-05

Protein inference from peptide identifications in shotgun proteomics must deal with ambiguities that arise due to the presence of peptides shared between different proteins, which is common in higher eukaryotes. Recently data independent acquisition (DIA) approaches have emerged as an alternative to the traditional data dependent acquisition (DDA) in shotgun proteomics experiments. MSE is the term used to name one of the DIA approaches used in QTOF instruments. MSE data require specialized software to process acquired spectra and to perform peptide and protein identifications. However the software available at the moment does not group the identified proteins in a transparent way by taking into account peptide evidence categories. Furthermore the inspection, comparison and report of the obtained results require tedious manual intervention. Here we report a software tool to address these limitations for MSE data. In this paper we present PAnalyzer, a software tool focused on the protein inference process of shotgun proteomics. Our approach considers all the identified proteins and groups them when necessary indicating their confidence using different evidence categories. PAnalyzer can read protein identification files in the XML output format of the ProteinLynx Global Server (PLGS) software provided by Waters Corporation for their MSE data, and also in the mzIdentML format recently standardized by HUPO-PSI. Multiple files can also be read simultaneously and are considered as technical replicates. Results are saved to CSV, HTML and mzIdentML (in the case of a single mzIdentML input file) files. An MSE analysis of a real sample is presented to compare the results of PAnalyzer and ProteinLynx Global Server. We present a software tool to deal with the ambiguities that arise in the protein inference process. Key contributions are support for MSE data analysis by ProteinLynx Global Server and technical replicates integration. PAnalyzer is an easy to use multiplatform and free software tool.

PAnalyzer: A software tool for protein inference in shotgun proteomics

PubMed Central

2012-01-01

Background Protein inference from peptide identifications in shotgun proteomics must deal with ambiguities that arise due to the presence of peptides shared between different proteins, which is common in higher eukaryotes. Recently data independent acquisition (DIA) approaches have emerged as an alternative to the traditional data dependent acquisition (DDA) in shotgun proteomics experiments. MSE is the term used to name one of the DIA approaches used in QTOF instruments. MSE data require specialized software to process acquired spectra and to perform peptide and protein identifications. However the software available at the moment does not group the identified proteins in a transparent way by taking into account peptide evidence categories. Furthermore the inspection, comparison and report of the obtained results require tedious manual intervention. Here we report a software tool to address these limitations for MSE data. Results In this paper we present PAnalyzer, a software tool focused on the protein inference process of shotgun proteomics. Our approach considers all the identified proteins and groups them when necessary indicating their confidence using different evidence categories. PAnalyzer can read protein identification files in the XML output format of the ProteinLynx Global Server (PLGS) software provided by Waters Corporation for their MSE data, and also in the mzIdentML format recently standardized by HUPO-PSI. Multiple files can also be read simultaneously and are considered as technical replicates. Results are saved to CSV, HTML and mzIdentML (in the case of a single mzIdentML input file) files. An MSE analysis of a real sample is presented to compare the results of PAnalyzer and ProteinLynx Global Server. Conclusions We present a software tool to deal with the ambiguities that arise in the protein inference process. Key contributions are support for MSE data analysis by ProteinLynx Global Server and technical replicates integration. PAnalyzer is an easy to use multiplatform and free software tool. PMID:23126499

What computational non-targeted mass spectrometry-based metabolomics can gain from shotgun proteomics.

PubMed

Hamzeiy, Hamid; Cox, Jürgen

2017-02-01

Computational workflows for mass spectrometry-based shotgun proteomics and untargeted metabolomics share many steps. Despite the similarities, untargeted metabolomics is lagging behind in terms of reliable fully automated quantitative data analysis. We argue that metabolomics will strongly benefit from the adaptation of successful automated proteomics workflows to metabolomics. MaxQuant is a popular platform for proteomics data analysis and is widely considered to be superior in achieving high precursor mass accuracies through advanced nonlinear recalibration, usually leading to five to ten-fold better accuracy in complex LC-MS/MS runs. This translates to a sharp decrease in the number of peptide candidates per measured feature, thereby strongly improving the coverage of identified peptides. We argue that similar strategies can be applied to untargeted metabolomics, leading to equivalent improvements in metabolite identification. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Recruiting Human Microbiome Shotgun Data to Site-Specific Reference Genomes

PubMed Central

Xie, Gary; Lo, Chien-Chi; Scholz, Matthew; Chain, Patrick S. G.

2014-01-01

The human body consists of innumerable multifaceted environments that predispose colonization by a number of distinct microbial communities, which play fundamental roles in human health and disease. In addition to community surveys and shotgun metagenomes that seek to explore the composition and diversity of these microbiomes, there are significant efforts to sequence reference microbial genomes from many body sites of healthy adults. To illustrate the utility of reference genomes when studying more complex metagenomes, we present a reference-based analysis of sequence reads generated from 55 shotgun metagenomes, selected from 5 major body sites, including 16 sub-sites. Interestingly, between 13% and 92% (62.3% average) of these shotgun reads were aligned to a then-complete list of 2780 reference genomes, including 1583 references for the human microbiome. However, no reference genome was universally found in all body sites. For any given metagenome, the body site-specific reference genomes, derived from the same body site as the sample, accounted for an average of 58.8% of the mapped reads. While different body sites did differ in abundant genera, proximal or symmetrical body sites were found to be most similar to one another. The extent of variation observed, both between individuals sampled within the same microenvironment, or at the same site within the same individual over time, calls into question comparative studies across individuals even if sampled at the same body site. This study illustrates the high utility of reference genomes and the need for further site-specific reference microbial genome sequencing, even within the already well-sampled human microbiome. PMID:24454771

A Bioinformatics Workflow for Variant Peptide Detection in Shotgun Proteomics*

PubMed Central

Li, Jing; Su, Zengliu; Ma, Ze-Qiang; Slebos, Robbert J. C.; Halvey, Patrick; Tabb, David L.; Liebler, Daniel C.; Pao, William; Zhang, Bing

2011-01-01

Shotgun proteomics data analysis usually relies on database search. However, commonly used protein sequence databases do not contain information on protein variants and thus prevent variant peptides and proteins from been identified. Including known coding variations into protein sequence databases could help alleviate this problem. Based on our recently published human Cancer Proteome Variation Database, we have created a protein sequence database that comprehensively annotates thousands of cancer-related coding variants collected in the Cancer Proteome Variation Database as well as noncancer-specific ones from the Single Nucleotide Polymorphism Database (dbSNP). Using this database, we then developed a data analysis workflow for variant peptide identification in shotgun proteomics. The high risk of false positive variant identifications was addressed by a modified false discovery rate estimation method. Analysis of colorectal cancer cell lines SW480, RKO, and HCT-116 revealed a total of 81 peptides that contain either noncancer-specific or cancer-related variations. Twenty-three out of 26 variants randomly selected from the 81 were confirmed by genomic sequencing. We further applied the workflow on data sets from three individual colorectal tumor specimens. A total of 204 distinct variant peptides were detected, and five carried known cancer-related mutations. Each individual showed a specific pattern of cancer-related mutations, suggesting potential use of this type of information for personalized medicine. Compatibility of the workflow has been tested with four popular database search engines including Sequest, Mascot, X!Tandem, and MyriMatch. In summary, we have developed a workflow that effectively uses existing genomic data to enable variant peptide detection in proteomics. PMID:21389108

[Injury patterns and roentgen findings in gunshot wounds with rare flint ammunition].

PubMed

Pollak, S; Lindermann, A

1990-01-01

Smoothbore shotgun barrels can fire cartridges with common pellet loads as well as shotgun slugs and rubber bullets. Other than conventional shot, the cylindrical Brenneke-type rifled shotgun slugs sometimes cause perforating wounds. The shotgun ammunition for use in self-defence can have a single projectile or several rubber pellets. Where the propellant is black powder, short range shots will probably leave searing marks and intensive soot deposits. Fired at close range, rubber bullets can penetrate through the skin into the body, fired at greater distance they cause contusions. A case of homicide (repeated firing with a 12-ga. pump gun) is used to present and discuss the injury patterns and X-ray findings after impact of Brenneke-type slugs and rubber bullets as well as of "classical" shot pellets.

Characterization of the Gut Microbiome Using 16S or Shotgun Metagenomics

PubMed Central

Jovel, Juan; Patterson, Jordan; Wang, Weiwei; Hotte, Naomi; O'Keefe, Sandra; Mitchel, Troy; Perry, Troy; Kao, Dina; Mason, Andrew L.; Madsen, Karen L.; Wong, Gane K.-S.

2016-01-01

The advent of next generation sequencing (NGS) has enabled investigations of the gut microbiome with unprecedented resolution and throughput. This has stimulated the development of sophisticated bioinformatics tools to analyze the massive amounts of data generated. Researchers therefore need a clear understanding of the key concepts required for the design, execution and interpretation of NGS experiments on microbiomes. We conducted a literature review and used our own data to determine which approaches work best. The two main approaches for analyzing the microbiome, 16S ribosomal RNA (rRNA) gene amplicons and shotgun metagenomics, are illustrated with analyses of libraries designed to highlight their strengths and weaknesses. Several methods for taxonomic classification of bacterial sequences are discussed. We present simulations to assess the number of sequences that are required to perform reliable appraisals of bacterial community structure. To the extent that fluctuations in the diversity of gut bacterial populations correlate with health and disease, we emphasize various techniques for the analysis of bacterial communities within samples (α-diversity) and between samples (β-diversity). Finally, we demonstrate techniques to infer the metabolic capabilities of a bacteria community from these 16S and shotgun data. PMID:27148170

Serum proteomic analysis identifies sex-specific differences in lipid metabolism and inflammation profiles in adults diagnosed with Asperger syndrome

PubMed Central

2014-01-01

Background The higher prevalence of Asperger Syndrome (AS) and other autism spectrum conditions in males has been known for many years. However, recent multiplex immunoassay profiling studies have shown that males and females with AS have distinct proteomic changes in serum. Methods Here, we analysed sera from adults diagnosed with AS (males = 14, females = 16) and controls (males = 13, females = 16) not on medication at the time of sample collection, using a combination of multiplex immunoassay and shotgun label-free liquid chromatography mass spectrometry (LC-MSE). The main objective was to identify sex-specific serum protein changes associated with AS. Results Multiplex immunoassay profiling led to identification of 16 proteins that were significantly altered in AS individuals in a sex-specific manner. Three of these proteins were altered in females (ADIPO, IgA, APOA1), seven were changed in males (BMP6, CTGF, ICAM1, IL-12p70, IL-16, TF, TNF-alpha) and six were changed in both sexes but in opposite directions (CHGA, EPO, IL-3, TENA, PAP, SHBG). Shotgun LC-MSE profiling led to identification of 13 serum proteins which had significant sex-specific changes in the AS group and, of these, 12 were altered in females (APOC2, APOE, ARMC3, CLC4K, FETUB, GLCE, MRRP1, PTPA, RN149, TLE1, TRIPB, ZC3HE) and one protein was altered in males (RGPD4). The free androgen index in females with AS showed an increased ratio of 1.63 compared to controls. Conclusion Taken together, the serum multiplex immunoassay and shotgun LC-MSE profiling results indicate that adult females with AS had alterations in proteins involved mostly in lipid transport and metabolism pathways, while adult males with AS showed changes predominantly in inflammation signalling. These results provide further evidence that the search for biomarkers or novel drug targets in AS may require stratification into male and female subgroups, and could lead to the development of novel targeted treatment approaches. PMID:24467795

Integrative proteomic analysis of the NMDA NR1 knockdown mouse model reveals effects on central and peripheral pathways associated with schizophrenia and autism spectrum disorders.

PubMed

Wesseling, Hendrik; Guest, Paul C; Lee, Chi-Ming; Wong, Erik Hf; Rahmoune, Hassan; Bahn, Sabine

2014-01-01

Over the last decade, the transgenic N-methyl-D-aspartate receptor (NMDAR) NR1-knockdown mouse (NR1(neo-/-)) has been investigated as a glutamate hypofunction model for schizophrenia. Recent research has now revealed that the model also recapitulates cognitive and negative symptoms in the continuum of other psychiatric diseases, particularly autism spectrum disorders (ASD). As previous studies have mostly focussed on behavioural readouts, a molecular characterisation of this model will help to identify novel biomarkers or potential drug targets. Here, we have used multiplex immunoassay analyses to investigate peripheral analyte alterations in serum of NR1(neo-/-) mice, as well as a combination of shotgun label-free liquid chromatography mass spectrometry, bioinformatic pathway analyses, and a shotgun-based 40-plex selected reaction monitoring (SRM) assay to investigate altered molecular pathways in the frontal cortex and hippocampus. All findings were cross compared to identify translatable findings between the brain and periphery. Multiplex immunoassay profiling led to identification of 29 analytes that were significantly altered in sera of NR1(neo-/-) mice. The highest magnitude changes were found for neurotrophic factors (VEGFA, EGF, IGF-1), apolipoprotein A1, and fibrinogen. We also found decreased levels of several chemokines. Following this, LC-MS(E) profiling led to identification of 48 significantly changed proteins in the frontal cortex and 41 in the hippocampus. In particular, MARCS, the mitochondrial pyruvate kinase, and CamKII-alpha were affected. Based on the combination of protein set enrichment and bioinformatic pathway analysis, we designed orthogonal SRM-assays which validated the abnormalities of proteins involved in synaptic long-term potentiation, myelination, and the ERK-signalling pathway in both brain regions. In contrast, increased levels of proteins involved in neurotransmitter metabolism and release were found only in the frontal cortex and abnormalities of proteins involved in the purinergic system were found exclusively in the hippocampus. Taken together, this multi-platform profiling study has identified peripheral changes which are potentially linked to central alterations in synaptic plasticity and neuronal function associated with NMDAR-NR1 hypofunction. Therefore, the reported proteomic changes may be useful as translational biomarkers in human and rodent model drug discovery efforts.

Protective ability of the standard U.S. Military Personal Armor System, Ground Troops (PASGT) fragmentation vest against common small arms projectiles.

PubMed

Roberts, G K; Bullian, M E

1993-08-01

This study analyzes the ballistic protection provided by the standard U.S. Military Personal Armor System, Ground Troops fragmentation vest against threats from handgun and shotgun projectiles. Damage assessment was conducted using 10% ordnance gelatin, a tissue simulant which has been proven to have a close correlation with living tissue. The standard fragmentation vest was shown to provide protection from commonly encountered handgun bullets and shotgun pellets, but not from shotgun slugs or center-fire rifle bullets.

Screening Currency Notes for Microbial Pathogens and Antibiotic Resistance Genes Using a Shotgun Metagenomic Approach

PubMed Central

Jalali, Saakshi; Kohli, Samantha; Latka, Chitra; Bhatia, Sugandha; Vellarikal, Shamsudheen Karuthedath; Sivasubbu, Sridhar; Scaria, Vinod; Ramachandran, Srinivasan

2015-01-01

Fomites are a well-known source of microbial infections and previous studies have provided insights into the sojourning microbiome of fomites from various sources. Paper currency notes are one of the most commonly exchanged objects and its potential to transmit pathogenic organisms has been well recognized. Approaches to identify the microbiome associated with paper currency notes have been largely limited to culture dependent approaches. Subsequent studies portrayed the use of 16S ribosomal RNA based approaches which provided insights into the taxonomical distribution of the microbiome. However, recent techniques including shotgun sequencing provides resolution at gene level and enable estimation of their copy numbers in the metagenome. We investigated the microbiome of Indian paper currency notes using a shotgun metagenome sequencing approach. Metagenomic DNA isolated from samples of frequently circulated denominations of Indian currency notes were sequenced using Illumina Hiseq sequencer. Analysis of the data revealed presence of species belonging to both eukaryotic and prokaryotic genera. The taxonomic distribution at kingdom level revealed contigs mapping to eukaryota (70%), bacteria (9%), viruses and archae (~1%). We identified 78 pathogens including Staphylococcus aureus, Corynebacterium glutamicum, Enterococcus faecalis, and 75 cellulose degrading organisms including Acidothermus cellulolyticus, Cellulomonas flavigena and Ruminococcus albus. Additionally, 78 antibiotic resistance genes were identified and 18 of these were found in all the samples. Furthermore, six out of 78 pathogens harbored at least one of the 18 common antibiotic resistance genes. To the best of our knowledge, this is the first report of shotgun metagenome sequence dataset of paper currency notes, which can be useful for future applications including as bio-surveillance of exchangeable fomites for infectious agents. PMID:26035208

Label-free shotgun proteomics and metabolite analysis reveal a significant metabolic shift during citrus fruit development

PubMed Central

Katz, Ehud; Boo, Kyung Hwan; Kim, Ho Youn; Eigenheer, Richard A.; Phinney, Brett S.; Shulaev, Vladimir; Negre-Zakharov, Florence; Sadka, Avi; Blumwald, Eduardo

2011-01-01

Label-free LC-MS/MS-based shot-gun proteomics was used to quantify the differential protein synthesis and metabolite profiling in order to assess metabolic changes during the development of citrus fruits. Our results suggested the occurrence of a metabolic change during citrus fruit maturation, where the organic acid and amino acid accumulation seen during the early stages of development shifted into sugar synthesis during the later stage of citrus fruit development. The expression of invertases remained unchanged, while an invertase inhibitor was up-regulated towards maturation. The increased expression of sucrose-phosphate synthase and sucrose-6-phosphate phosphatase and the rapid sugar accumulation suggest that sucrose is also being synthesized in citrus juice sac cells during the later stage of fruit development. PMID:21841177

Shotgun mitogenomics across body size classes in a local assemblage of tropical Diptera: Phylogeny, species diversity and mitochondrial abundance spectrum.

PubMed

Choo, Le Qin; Crampton-Platt, Alex; Vogler, Alfried P

2017-10-01

Mitochondrial genomes can be assembled readily from shotgun-sequenced DNA mixtures of mass-trapped arthropods ("mitochondrial metagenomics"), speeding up the taxonomic characterization. Bulk sequencing was conducted on some 800 individuals of Diptera obtained by canopy fogging of a single tree in Borneo dominated by small (<1.5 mm) individuals. Specimens were split into five body size classes for DNA extraction, to equalize read numbers across specimens and to study how body size, a key ecological trait, interacts with species and phylogenetic diversity. Genome assembly produced 304 orthologous mitochondrial contigs presumed to each represent a different species. The small-bodied fraction was the by far most species-rich (187 contigs). Identification of contigs was through phylogenetic analysis together with 56 reference mitogenomes, which placed most of the Bornean community into seven clades of small-bodied species, indicating phylogenetic conservation of body size. Mapping of shotgun reads against the mitogenomes showed wide ranges of read abundances within each size class. Ranked read abundance plots were largely log-linear, indicating a uniformly filled abundance spectrum, especially for small-bodied species. Small-bodied species differed greatly from other size classes in neutral metacommunity parameters, exhibiting greater levels of immigration, besides greater total community size. We suggest that the established uses of mitochondrial metagenomics for analysis of species and phylogenetic diversity can be extended to parameterize recent theories of community ecology and biodiversity, and by focusing on the number mitochondria, rather than individuals, a new theoretical framework for analysis of mitochondrial abundance spectra can be developed that incorporates metabolic activity approximated by the count of mitochondria. © 2017 John Wiley & Sons Ltd.

Bioaffinity magnetic reactor for peptide digestion followed by analysis using bottom-up shotgun proteomics strategy.

PubMed

Korecká, Lucie; Jankovicová, Barbora; Krenková, Jana; Hernychová, Lenka; Slováková, Marcela; Le-Nell, Anne; Chmelik, Josef; Foret, Frantisek; Viovy, Jean-Louis; Bilková, Zusana

2008-02-01

We report an efficient and streamlined way to improve the analysis and identification of peptides and proteins in complex mixtures of soluble proteins, cell lysates, etc. By using the shotgun proteomics methodology combined with bioaffinity purification we can remove or minimize the interference contamination of a complex tryptic digest and so avoid the time-consuming separation steps before the final MS analysis. We have proved that by means of enzymatic fragmentation (endoproteinases with Arg-C or/and Lys-C specificity) connected with the isolation of specific peptides we can obtain a simplified peptide mixture for easier identification of the entire protein. A new bioaffinity sorbent was developed for this purpose. Anhydrotrypsin (AHT), an inactive form of trypsin with an affinity for peptides with arginine (Arg) or lysine (Lys) at the C-terminus, was immobilized onto micro/nanoparticles with superparamagnetic properties (silica magnetite particles (SiMAG)-Carboxyl, Chemicell, Germany). This AHT carrier with a determined binding capacity (26.8 nmol/mg of carrier) was tested with a model peptide, human neurotensin, and the resulting MS spectra confirmed the validity of this approach.

Approaches to the management of shotgun injuries.

PubMed

Flint, L M; Cryer, H M; Howard, D A; Richardson, J D

1984-05-01

Shotgun wounds present specific challenges for the surgeon. Multiple penetrating wounds frequently involve large anatomic areas with potential multi-system injury. Experience with 121 patients sustaining shotgun wounds over the 5-year period ending 31 December 1981 was reviewed to assess results and evaluate treatment protocols. Sixty-six patients had chest wounds with pleural penetration. Twenty-four wounds were minor and were observed. Each had less than five pellets penetrating the pleura. Twenty-two patients had close-range injuries. Fourteen of these required chest tube drainage alone and eight patients required thoracotomy for control of bleeding. Eleven patients died, six as a direct result of the chest injury. In 55 patients with abdominal-retroperitoneal wounds exploratory operations were done if more than four pellets were thought to be lodged intraperitoneally or if signs of peritonitis were present, while lesser wounds without peritoneal findings were observed. In the 15 patients who did not have exploratory operations, there were no deaths or major complications. Thirty-five patients had exploratory operations. Two patients had five intraperitoneal missiles and no clinical evidence of peritonitis but were found to have significant intestinal perforations. Four patients died. Eighty-three patients with extremity wounds were classified according to location of injury. Forty-five had upper extremity wounds, with nine vascular injuries. Two patients died and one limb was amputated because of soft tissue infection. Thirty-eight patients had lower extremity wounds. Five had major vascular injuries. Preoperative arteriography was obtained in 13 patients with extremity injuries; the results of one of these were falsely negative. There were no deaths or amputations.(ABSTRACT TRUNCATED AT 250 WORDS)

Comprehensive genomic analysis of a plant growth-promoting rhizobacterium Pantoea agglomerans strain P5.

PubMed

Shariati J, Vahid; Malboobi, Mohammad Ali; Tabrizi, Zeinab; Tavakol, Elahe; Owilia, Parviz; Safari, Maryam

2017-11-15

In this study, we provide a comparative genomic analysis of Pantoea agglomerans strain P5 and 10 closely related strains based on phylogenetic analyses. A next-generation shotgun strategy was implemented using the Illumina HiSeq 2500 technology followed by core- and pan-genome analysis. The genome of P. agglomerans strain P5 contains an assembly size of 5082485 bp with 55.4% G + C content. P. agglomerans consists of 2981 core and 3159 accessory genes for Coding DNA Sequences (CDSs) based on the pan-genome analysis. Strain P5 can be grouped closely with strains PG734 and 299 R using pan and core genes, respectively. All the predicted and annotated gene sequences were allocated to KEGG pathways. Accordingly,  genes involved in plant growth-promoting (PGP) ability, including phosphate solubilization, IAA and siderophore production, acetoin and 2,3-butanediol synthesis and bacterial secretion, were assigned. This study provides an in-depth view of the PGP characteristics of strain P5, highlighting its potential use in agriculture as a biofertilizer.

Enrichment dynamics of Listeria monocytogenes and the associated microbiome from naturally contaminated ice cream linked to a listeriosis outbreak.

PubMed

Ottesen, Andrea; Ramachandran, Padmini; Reed, Elizabeth; White, James R; Hasan, Nur; Subramanian, Poorani; Ryan, Gina; Jarvis, Karen; Grim, Christopher; Daquiqan, Ninalynn; Hanes, Darcy; Allard, Marc; Colwell, Rita; Brown, Eric; Chen, Yi

2016-11-16

Microbiota that co-enrich during efforts to recover pathogens from foodborne outbreaks interfere with efficient detection and recovery. Here, dynamics of co-enriching microbiota during recovery of Listeria monocytogenes from naturally contaminated ice cream samples linked to an outbreak are described for three different initial enrichment formulations used by the Food and Drug Administration (FDA), the International Organization of Standardization (ISO), and the United States Department of Agriculture (USDA). Enrichment cultures were analyzed using DNA extraction and sequencing from samples taken every 4 h throughout 48 h of enrichment. Resphera Insight and CosmosID analysis tools were employed for high-resolution profiling of 16S rRNA amplicons and whole genome shotgun data, respectively. During enrichment, other bacterial taxa were identified, including Anoxybacillus, Geobacillus, Serratia, Pseudomonas, Erwinia, and Streptococcus spp. Surprisingly, incidence of L. monocytogenes was proportionally greater at hour 0 than when tested 4, 8, and 12 h later with all three enrichment schemes. The corresponding increase in Anoxybacillus and Geobacillus spp.indicated these taxa co-enriched in competition with L. monocytogenes during early enrichment hours. L. monocytogenes became dominant after 24 h in all three enrichments. DNA sequences obtained from shotgun metagenomic data of Listeria monocytogenes at 48 h were assembled to produce a consensus draft genome which appeared to have a similar tracking utility to pure culture isolates of L. monocytogenes. All three methods performed equally well for enrichment of Listeria monocytogenes. The observation of potential competitive exclusion of L. mono by Anoxybacillus and Geobacillus in early enrichment hours provided novel information that may be used to further optimize enrichment formulations. Application of Resphera Insight for high-resolution analysis of 16S amplicon sequences accurately identified L. monocytogenes. Both shotgun and 16S rRNA data supported the presence of three slightly variable genomes of L. monocytogenes. Moreover, the draft assembly of a consensus genome of L. monocytogenes from shotgun metagenomic data demonstrated the potential utility of this approach to expedite trace-back of outbreak-associated strains, although further validation will be needed to confirm this utility.

Low-pass sequencing for microbial comparative genomics

PubMed Central

Goo, Young Ah; Roach, Jared; Glusman, Gustavo; Baliga, Nitin S; Deutsch, Kerry; Pan, Min; Kennedy, Sean; DasSarma, Shiladitya; Victor Ng, Wailap; Hood, Leroy

2004-01-01

Background We studied four extremely halophilic archaea by low-pass shotgun sequencing: (1) the metabolically versatile Haloarcula marismortui; (2) the non-pigmented Natrialba asiatica; (3) the psychrophile Halorubrum lacusprofundi and (4) the Dead Sea isolate Halobaculum gomorrense. Approximately one thousand single pass genomic sequences per genome were obtained. The data were analyzed by comparative genomic analyses using the completed Halobacterium sp. NRC-1 genome as a reference. Low-pass shotgun sequencing is a simple, inexpensive, and rapid approach that can readily be performed on any cultured microbe. Results As expected, the four archaeal halophiles analyzed exhibit both bacterial and eukaryotic characteristics as well as uniquely archaeal traits. All five halophiles exhibit greater than sixty percent GC content and low isoelectric points (pI) for their predicted proteins. Multiple insertion sequence (IS) elements, often involved in genome rearrangements, were identified in H. lacusprofundi and H. marismortui. The core biological functions that govern cellular and genetic mechanisms of H. sp. NRC-1 appear to be conserved in these four other halophiles. Multiple TATA box binding protein (TBP) and transcription factor IIB (TFB) homologs were identified from most of the four shotgunned halophiles. The reconstructed molecular tree of all five halophiles shows a large divergence between these species, but with the closest relationship being between H. sp. NRC-1 and H. lacusprofundi. Conclusion Despite the diverse habitats of these species, all five halophiles share (1) high GC content and (2) low protein isoelectric points, which are characteristics associated with environmental exposure to UV radiation and hypersalinity, respectively. Identification of multiple IS elements in the genome of H. lacusprofundi and H. marismortui suggest that genome structure and dynamic genome reorganization might be similar to that previously observed in the IS-element rich genome of H. sp. NRC-1. Identification of multiple TBP and TFB homologs in these four halophiles are consistent with the hypothesis that different types of complex transcriptional regulation may occur through multiple TBP-TFB combinations in response to rapidly changing environmental conditions. Low-pass shotgun sequence analyses of genomes permit extensive and diverse analyses, and should be generally useful for comparative microbial genomics. PMID:14718067

Effects of Fe and Mn deficiencies on the protein profiles of tomato (Solanum lycopersicum) xylem sap as revealed by shotgun analyses.

PubMed

Ceballos-Laita, Laura; Gutierrez-Carbonell, Elain; Takahashi, Daisuke; Abadía, Anunciación; Uemura, Matsuo; Abadía, Javier; López-Millán, Ana Flor

2018-01-06

The aim of this work was to study the effects of Fe and Mn deficiencies on the xylem sap proteome of tomato using a shotgun proteomic approach, with the final goal of elucidating plant response mechanisms to these stresses. This approach yielded 643 proteins reliably identified and quantified with 70% of them predicted as secretory. Iron and Mn deficiencies caused statistically significant and biologically relevant abundance changes in 119 and 118 xylem sap proteins, respectively. In both deficiencies, metabolic pathways most affected were protein metabolism, stress/oxidoreductases and cell wall modifications. First, results suggest that Fe deficiency elicited more stress responses than Mn deficiency, based on the changes in oxidative and proteolytic enzymes. Second, both nutrient deficiencies affect the secondary cell wall metabolism, with changes in Fe deficiency occurring via peroxidase activity, and in Mn deficiency involving peroxidase, Cu-oxidase and fasciclin-like arabinogalactan proteins. Third, the primary cell wall metabolism was affected by both nutrient deficiencies, with changes following opposite directions as judged from the abundances of several glycoside-hydrolases with endo-glycolytic activities and pectin esterases. Fourth, signaling pathways via xylem involving CLE and/or lipids as well as changes in phosphorylation and N-glycosylation also play a role in the responses to these stresses. Biological significance In spite of being essential for the delivery of nutrients to the shoots, our knowledge of xylem responses to nutrient deficiencies is very limited. The present work applies a shotgun proteomic approach to unravel the effects of Fe and Mn deficiencies on the xylem sap proteome. Overall, Fe deficiency seems to elicit more stress in the xylem sap proteome than Mn deficiency, based on the changes measured in proteolytic and oxido-reductase proteins, whereas both nutrients exert modifications in the composition of the primary and secondary cell wall. Cell wall modifications could affect the mechanical and permeability properties of the xylem sap vessels, and therefore ultimately affect solute transport and distribution to the leaves. Results also suggest that signaling cascades involving lipid and peptides might play a role in nutrient stress signaling and pinpoint interesting candidates for future studies. Finally, both nutrient deficiencies seem to affect phosphorylation and glycosylation processes, again following an opposite pattern. Copyright © 2017 Elsevier B.V. All rights reserved.

Metaproteogenomics Reveals Taxonomic and Functional Changes between Cecal and Fecal Microbiota in Mouse

PubMed Central

Tanca, Alessandro; Manghina, Valeria; Fraumene, Cristina; Palomba, Antonio; Abbondio, Marcello; Deligios, Massimo; Silverman, Michael; Uzzau, Sergio

2017-01-01

Previous studies on mouse models report that cecal and fecal microbial communities may differ in the taxonomic structure, but little is known about their respective functional activities. Here, we employed a metaproteogenomic approach, including 16S rRNA gene sequencing, shotgun metagenomics and shotgun metaproteomics, to analyze the microbiota of paired mouse cecal contents (CCs) and feces, with the aim of identifying changes in taxon-specific functions. As a result, Gram-positive anaerobes were observed as considerably higher in CCs, while several key enzymes, involved in oxalate degradation, glutamate/glutamine metabolism, and redox homeostasis, and most actively expressed by Bacteroidetes, were clearly more represented in feces. On the whole, taxon and function abundance appeared to vary consistently with environmental changes expected to occur throughout the transit from the cecum to outside the intestine, especially when considering metaproteomic data. The results of this study indicate that functional and metabolic differences exist between CC and stool samples, paving the way to further metaproteogenomic investigations aimed at elucidating the functional dynamics of the intestinal microbiota. PMID:28352255

Replica Exchange Improves Sampling in Low-Resolution Docking Stage of RosettaDock

PubMed Central

Zhang, Zhe; Lange, Oliver F.

2013-01-01

Many protein-protein docking protocols are based on a shotgun approach, in which thousands of independent random-start trajectories minimize the rigid-body degrees of freedom. Another strategy is enumerative sampling as used in ZDOCK. Here, we introduce an alternative strategy, ReplicaDock, using a small number of long trajectories of temperature replica exchange. We compare replica exchange sampling as low-resolution stage of RosettaDock with RosettaDock's original shotgun sampling as well as with ZDOCK. A benchmark of 30 complexes starting from structures of the unbound binding partners shows improved performance for ReplicaDock and ZDOCK when compared to shotgun sampling at equal or less computational expense. ReplicaDock and ZDOCK consistently reach lower energies and generate significantly more near-native conformations than shotgun sampling. Accordingly, they both improve typical metrics of prediction quality of complex structures after refinement. Additionally, the refined ReplicaDock ensembles reach significantly lower interface energies and many previously hidden features of the docking energy landscape become visible when ReplicaDock is applied. PMID:24009670

Proteomic analysis of Chlorella vulgaris: Potential targets for enhanced lipid accumulation

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

Guarnieri, Michael T.; Nag, Ambarish; Yang, Shihui

2013-11-01

Oleaginous microalgae are capable of producing large quantities of fatty acids and triacylglycerides. As such, they are promising feedstocks for the production of biofuels and bioproducts. Genetic strain-engineering strategies offer a means to accelerate the commercialization of algal biofuels by improving the rate and total accumulation of microalgal lipids. However, the industrial potential of these organisms remains to be met, largely due to the incomplete knowledgebase surrounding the mechanisms governing the induction of algal lipid biosynthesis. Such strategies require further elucidation of genes and gene products controlling algal lipid accumulation. In this study, we have set out to examine thesemore » mechanisms and identify novel strain-engineering targets in the oleaginous microalga, Chlorella vulgaris. Comparative shotgun proteomic analyses have identified a number of novel targets, including previously unidentified transcription factors and proteins involved in cell signaling and cell cycle regulation. These results lay the foundation for strain-improvement strategies and demonstrate the power of translational proteomic analysis.« less

Deeper insight into the structure of the anaerobic digestion microbial community; the biogas microbiome database is expanded with 157 new genomes.

PubMed

Treu, Laura; Kougias, Panagiotis G; Campanaro, Stefano; Bassani, Ilaria; Angelidaki, Irini

2016-09-01

This research aimed to better characterize the biogas microbiome by means of high throughput metagenomic sequencing and to elucidate the core microbial consortium existing in biogas reactors independently from the operational conditions. Assembly of shotgun reads followed by an established binning strategy resulted in the highest, up to now, extraction of microbial genomes involved in biogas producing systems. From the 236 extracted genome bins, it was remarkably found that the vast majority of them could only be characterized at high taxonomic levels. This result confirms that the biogas microbiome is comprised by a consortium of unknown species. A comparative analysis between the genome bins of the current study and those extracted from a previous metagenomic assembly demonstrated a similar phylogenetic distribution of the main taxa. Finally, this analysis led to the identification of a subset of common microbes that could be considered as the core essential group in biogas production. Copyright © 2016 Elsevier Ltd. All rights reserved.

Secretome analysis of the thermophilic xylanase hyper-producer Thermomyces lanuginosus SSBP cultivated on corn cobs.

PubMed

Winger, A M; Heazlewood, J L; Chan, L J G; Petzold, C J; Permaul, K; Singh, S

2014-11-01

Thermomyces lanuginosus is a thermophilic fungus known for its ability to produce industrially important enzymes including large amounts of xylanase, the key enzyme in hemicellulose hydrolysis. The secretome of T. lanuginosus SSBP was profiled by shotgun proteomics to elucidate important enzymes involved in hemicellulose saccharification and to characterise the presence of other industrially interesting enzymes. This study reproducibly identified a total of 74 proteins in the supernatant following growth on corn cobs. An analysis of proteins revealed nine glycoside hydrolase (GH) enzymes including xylanase GH11, β-xylosidase GH43, β-glucosidase GH3, α-galactosidase GH36 and trehalose hydrolase GH65. Two commercially produced Thermomyces enzymes, lipase and amylase, were also identified. In addition, other industrially relevant enzymes not currently explored in Thermomyces were identified including glutaminase, fructose-bisphosphate aldolase and cyanate hydratase. Overall, these data provide insight into the novel ability of a cellulase-free fungus to utilise lignocellulosic material, ultimately producing a number of enzymes important to various industrial processes.

Missing Value Monitoring Enhances the Robustness in Proteomics Quantitation.

PubMed

Matafora, Vittoria; Corno, Andrea; Ciliberto, Andrea; Bachi, Angela

2017-04-07

In global proteomic analysis, it is estimated that proteins span from millions to less than 100 copies per cell. The challenge of protein quantitation by classic shotgun proteomic techniques relies on the presence of missing values in peptides belonging to low-abundance proteins that lowers intraruns reproducibility affecting postdata statistical analysis. Here, we present a new analytical workflow MvM (missing value monitoring) able to recover quantitation of missing values generated by shotgun analysis. In particular, we used confident data-dependent acquisition (DDA) quantitation only for proteins measured in all the runs, while we filled the missing values with data-independent acquisition analysis using the library previously generated in DDA. We analyzed cell cycle regulated proteins, as they are low abundance proteins with highly dynamic expression levels. Indeed, we found that cell cycle related proteins are the major components of the missing values-rich proteome. Using the MvM workflow, we doubled the number of robustly quantified cell cycle related proteins, and we reduced the number of missing values achieving robust quantitation for proteins over ∼50 molecules per cell. MvM allows lower quantification variance among replicates for low abundance proteins with respect to DDA analysis, which demonstrates the potential of this novel workflow to measure low abundance, dynamically regulated proteins.

Comparative Proteomic Analysis of Tolerance and Adaptation of Ethanologenic Saccharomyces cerevisiae to Furfural, a Lignocellulosic Inhibitory Compound▿ †

PubMed Central

Lin, Feng-Ming; Qiao, Bin; Yuan, Ying-Jin

2009-01-01

The molecular mechanism involved in tolerance and adaptation of ethanologenic Saccharomyces cerevisiae to inhibitors (such as furfural, acetic acid, and phenol) represented in lignocellulosic hydrolysate is still unclear. Here, 18O-labeling-aided shotgun comparative proteome analysis was applied to study the global protein expression profiles of S. cerevisiae under conditions of treatment of furfural compared with furfural-free fermentation profiles. Proteins involved in glucose fermentation and/or the tricarboxylic acid cycle were upregulated in cells treated with furfural compared with the control cells, while proteins involved in glycerol biosynthesis were downregulated. Differential levels of expression of alcohol dehydrogenases were observed. On the other hand, the levels of NADH, NAD+, and NADH/NAD+ were reduced whereas the levels of ATP and ADP were increased. These observations indicate that central carbon metabolism, levels of alcohol dehydrogenases, and the redox balance may be related to tolerance of ethanologenic yeast for and adaptation to furfural. Furthermore, proteins involved in stress response, including the unfolded protein response, oxidative stress, osmotic and salt stress, DNA damage and nutrient starvation, were differentially expressed, a finding that was validated by quantitative real-time reverse transcription-PCR to further confirm that the general stress responses are essential for cellular defense against furfural. These insights into the response of yeast to the presence of furfural will benefit the design and development of inhibitor-tolerant ethanologenic yeast by metabolic engineering or synthetic biology. PMID:19363068

75 FR 52024 - Agency Information Collection Activities: Proposed Collection; Comments Requested

Federal Register 2010, 2011, 2012, 2013, 2014

2010-08-24

... of information collection under review: Letter Application to Obtain Authorization for the Assembly... Application to Obtain Authorization for the Assembly of a Nonsporting Rifle or Nonsporting Shotgun for the... provide a means to obtain authorization for the assembly of a nonsporting rifle or nonsporting shotgun for...

Effect of MK-801 and Clozapine on the Proteome of Cultured Human Oligodendrocytes

PubMed Central

Cassoli, Juliana S.; Iwata, Keiko; Steiner, Johann; Guest, Paul C.; Turck, Christoph W.; Nascimento, Juliana M.; Martins-de-Souza, Daniel

2016-01-01

Separate lines of evidence have demonstrated the involvement of N-methyl-D-aspartate (NMDA) receptor and oligodendrocyte dysfunctions in schizophrenia. Here, we have carried out shotgun mass spectrometry proteome analysis of oligodendrocytes treated with the NMDA receptor antagonist MK-801 to gain potential insights into these effects at the molecular level. The MK-801 treatment led to alterations in the levels of 68 proteins, which are associated with seven distinct biological processes. Most of these proteins are involved in energy metabolism and many have been found to be dysregulated in previous proteomic studies of post-mortem brain tissues from schizophrenia patients. Finally, addition of the antipsychotic clozapine to MK-801-treated oligodendrocyte cultures resulted in changes in the levels of 45 proteins and treatment with clozapine alone altered 122 proteins and many of these showed opposite changes to the MK-801 effects. Therefore, these proteins and the associated energy metabolism pathways should be explored as potential biomarkers of antipsychotic efficacy. In conclusion, MK-801 treatment of oligodendrocytes may provide a useful model for testing the efficacy of novel treatment approaches. PMID:26973466

Metal dust deposition in a shotgun wound associated with barrel modification.

PubMed

Williams, Andrew S; Bowes, Matthew J

2016-03-01

Contact-range gunshot wounds commonly demonstrate deposition of black soot in and around the wound. Deposition of other visible pigments originating from the firearm has not been specifically described. In the current case, an adult male was found dead adjacent to a shotgun fixed in a vice grip with a modified, shortened barrel. A handheld, powered, metal grinding wheel was nearby. Autopsy revealed an intraoral gunshot wound, including soot deposition in and around the mouth and within the wound track. In addition, there was a peculiar, gray, lustrous film on the lips, gingiva, and anterior teeth. The material was concentrated around the most severe areas of injury in the anterior mouth and easily rubbed off with a cotton swab. It was not visualized in the rest of the mouth and not present in the larynx, or the esophagus. Overall, our opinion is that this unique, gray, lustrous film represents deposition of fine metallic dust that accumulated in the barrel of the shotgun during its modification with the grinding wheel. This type of unique pigment deposition should be recognized by forensic pathologists as possibly being related to the discharge of a firearm with a recently modified barrel or other cause for fine metallic dust accumulation within the barrel. Depending on the circumstances of the case, collection of samples of such metal dust deposits could be indicated for subsequent analysis.

Automated and Accurate Estimation of Gene Family Abundance from Shotgun Metagenomes

PubMed Central

Nayfach, Stephen; Bradley, Patrick H.; Wyman, Stacia K.; Laurent, Timothy J.; Williams, Alex; Eisen, Jonathan A.; Pollard, Katherine S.; Sharpton, Thomas J.

2015-01-01

Shotgun metagenomic DNA sequencing is a widely applicable tool for characterizing the functions that are encoded by microbial communities. Several bioinformatic tools can be used to functionally annotate metagenomes, allowing researchers to draw inferences about the functional potential of the community and to identify putative functional biomarkers. However, little is known about how decisions made during annotation affect the reliability of the results. Here, we use statistical simulations to rigorously assess how to optimize annotation accuracy and speed, given parameters of the input data like read length and library size. We identify best practices in metagenome annotation and use them to guide the development of the Shotgun Metagenome Annotation Pipeline (ShotMAP). ShotMAP is an analytically flexible, end-to-end annotation pipeline that can be implemented either on a local computer or a cloud compute cluster. We use ShotMAP to assess how different annotation databases impact the interpretation of how marine metagenome and metatranscriptome functional capacity changes across seasons. We also apply ShotMAP to data obtained from a clinical microbiome investigation of inflammatory bowel disease. This analysis finds that gut microbiota collected from Crohn’s disease patients are functionally distinct from gut microbiota collected from either ulcerative colitis patients or healthy controls, with differential abundance of metabolic pathways related to host-microbiome interactions that may serve as putative biomarkers of disease. PMID:26565399

Longitudinal Metagenomic Analysis of Hospital Air Identifies Clinically Relevant Microbes.

PubMed

King, Paula; Pham, Long K; Waltz, Shannon; Sphar, Dan; Yamamoto, Robert T; Conrad, Douglas; Taplitz, Randy; Torriani, Francesca; Forsyth, R Allyn

2016-01-01

We describe the sampling of sixty-three uncultured hospital air samples collected over a six-month period and analysis using shotgun metagenomic sequencing. Our primary goals were to determine the longitudinal metagenomic variability of this environment, identify and characterize genomes of potential pathogens and determine whether they are atypical to the hospital airborne metagenome. Air samples were collected from eight locations which included patient wards, the main lobby and outside. The resulting DNA libraries produced 972 million sequences representing 51 gigabases. Hierarchical clustering of samples by the most abundant 50 microbial orders generated three major nodes which primarily clustered by type of location. Because the indoor locations were longitudinally consistent, episodic relative increases in microbial genomic signatures related to the opportunistic pathogens Aspergillus, Penicillium and Stenotrophomonas were identified as outliers at specific locations. Further analysis of microbial reads specific for Stenotrophomonas maltophilia indicated homology to a sequenced multi-drug resistant clinical strain and we observed broad sequence coverage of resistance genes. We demonstrate that a shotgun metagenomic sequencing approach can be used to characterize the resistance determinants of pathogen genomes that are uncharacteristic for an otherwise consistent hospital air microbial metagenomic profile.

Application of an Improved Proteomics Method for Abundant Protein Cleanup: Molecular and Genomic Mechanisms Study in Plant Defense*

PubMed Central

Zhang, Yixiang; Gao, Peng; Xing, Zhuo; Jin, Shumei; Chen, Zhide; Liu, Lantao; Constantino, Nasie; Wang, Xinwang; Shi, Weibing; Yuan, Joshua S.; Dai, Susie Y.

2013-01-01

High abundance proteins like ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) impose a consistent challenge for the whole proteome characterization using shot-gun proteomics. To address this challenge, we developed and evaluated Polyethyleneimine Assisted Rubisco Cleanup (PARC) as a new method by combining both abundant protein removal and fractionation. The new approach was applied to a plant insect interaction study to validate the platform and investigate mechanisms for plant defense against herbivorous insects. Our results indicated that PARC can effectively remove Rubisco, improve the protein identification, and discover almost three times more differentially regulated proteins. The significantly enhanced shot-gun proteomics performance was translated into in-depth proteomic and molecular mechanisms for plant insect interaction, where carbon re-distribution was used to play an essential role. Moreover, the transcriptomic validation also confirmed the reliability of PARC analysis. Finally, functional studies were carried out for two differentially regulated genes as revealed by PARC analysis. Insect resistance was induced by over-expressing either jacalin-like or cupin-like genes in rice. The results further highlighted that PARC can serve as an effective strategy for proteomics analysis and gene discovery. PMID:23943779

Shotgun Optical Maps of the Whole Escherichia coli O157:H7 Genome

PubMed Central

Lim, Alex; Dimalanta, Eileen T.; Potamousis, Konstantinos D.; Yen, Galex; Apodoca, Jennifer; Tao, Chunhong; Lin, Jieyi; Qi, Rong; Skiadas, John; Ramanathan, Arvind; Perna, Nicole T.; Plunkett, Guy; Burland, Valerie; Mau, Bob; Hackett, Jeremiah; Blattner, Frederick R.; Anantharaman, Thomas S.; Mishra, Bhubaneswar; Schwartz, David C.

2001-01-01

We have constructed NheI and XhoI optical maps of Escherichia coli O157:H7 solely from genomic DNA molecules to provide a uniquely valuable scaffold for contig closure and sequence validation. E. coli O157:H7 is a common pathogen found in contaminated food and water. Our approach obviated the need for the analysis of clones, PCR products, and hybridizations, because maps were constructed from ensembles of single DNA molecules. Shotgun sequencing of bacterial genomes remains labor-intensive, despite advances in sequencing technology. This is partly due to manual intervention required during the last stages of finishing. The applicability of optical mapping to this problem was enhanced by advances in machine vision techniques that improved mapping throughput and created a path to full automation of mapping. Comparisons were made between maps and sequence data that characterized sequence gaps and guided nascent assemblies. PMID:11544203

Identification of the Viridicatumtoxin and Griseofulvin Gene Clusters from Penicillium aethiopicum

PubMed Central

Chooi, Yit-Heng; Cacho, Ralph; Tang, Yi

2010-01-01

SUMMARY Penicillium aethiopicum produces two structurally interesting and biologically active polyketides: the tetracycline-like viridicatumtoxin 1 and the classic antifungal agent griseofulvin 2. Here, we report the concurrent discovery of the two corresponding biosynthetic gene clusters (vrt and gsf) by 454 shotgun sequencing. Gene deletions confirmed two nonreducing PKSs (NRPKS), vrtA and gsfA, are required for the biosynthesis of 1 and 2, respectively. Both PKSs share similar domain architectures and lack a C-terminal thioesterase domain. We identified gsfI as the chlorinase involved in the biosynthesis of 2, as deletion of gsfI resulted in the accumulation of decholorogriseofulvin 3. Comparative analysis with the P. chrysogenum genome revealed that both clusters are embedded within conserved syntenic regions of P. aethiopicum chromosomes. Discovery of the vrt and gsf clusters provided the basis for genetic and biochemical studies of the pathways. PMID:20534346

The genome of black cottonwood, Populus trichocarpa (Torr. & Gray)

Treesearch

G.A. Tuskan; S. DiFazio; S. Jansson; J. Bohlmann; I. Grigoriev; U. Hellsten; N. Putnam; S. Ralph; S. Rombauts; A. Salamov; J. Schein; L. Sterck; A. Aerts; R.R. Bhalerao; R.P. Bhalerao; D. Blaudez; W. Boerjan; A. Brun; A. Brunner; V. Busov; M. Campbell; J. Carlson; M. Chalot; J. Chapman; G.-L. Chen; D. Cooper; P.M. Coutinho; J. Couturier; S. Covert; Q. Cronk; R. Cunningham; J. Davis; S. Degroeve; A. Dejardin; C. dePamphilis; J. Detter; B. Dirks; U. Dubchak; S. Duplessis; J. Ehlting; B. Ellis; K. Gendler; D. Goodstein; M. Gribskov; J. Grimwood; A. Groover; L. Gunter; B. Hamberger; B. Heinze; Y. Helariutta; B. Henrissat; D. Holligan; R. Holt; W. Huang; N. Islam-Faridi; S. Jones; M. Jones-Rhoades; R. Jorgensen; C. Joshi; J. Kangasjarvi; J. Karlsson; C. Kelleher; R. Kirkpatrick; M. Kirst; A. Kohler; U. Kalluri; F. Larimer; J. Leebens-Mack; J.-C. Leple; P. Locascio; Y. Lou; S. Lucas; F. Martin; B. Montanini; C. Napoli; D.R. Nelson; C. Nelson; K. Nieminen; O. Nilsson; V. Pereda; G. Peter; R. Philippe; G. Pilate; A. Poliakov; J. Razumovskaya; P. Richardson; C. Rinaldi; K. Ritland; P. Rouze; D. Ryaboy; J. Schumtz; J. Schrader; B. Segerman; H. Shin; A. Siddiqui; F. Sterky; A. Terry; C.-J. Tsai; E. Uberbacher; P. Unneberg; J. Vahala; K. Wall; S. Wessler; G. Yang; T. Yin; C. Douglas; M. Marra; G. Sandberg; Y. Van de Peer; D. Rokhsar

2006-01-01

We report the draft genome of the black cottonwood tree, Populus trichocarpa. Integration of shotgun sequence assembly with genetic mapping enabled chromosome-scale reconstruction of the genome. More than 45,000 putative protein-coding genes were identified. Analysis of the assembled genome revealed a whole-genome duplication event; about 8000 pairs...

27 CFR 478.11 - Meaning of terms.

Code of Federal Regulations, 2012 CFR

2012-04-01

... than one shot, without manual reloading, by a single function of the trigger. The term shall also... than an antique firearm. The term shall not include (a) any shotgun shot or pellet not designed for use as the single, complete projectile load for one shotgun hull or casing, nor (b) any unloaded, non...

27 CFR 479.11 - Meaning of terms.

Code of Federal Regulations, 2014 CFR

2014-04-01

.... Any other weapon. Any weapon or device capable of being concealed on the person from which a shot can...) missile having an explosive or incendiary charge of more than one-quarter ounce, (5) mine, or (6) similar... have a bore of more than one-half inch in diameter, except a shotgun or shotgun shell which the...

27 CFR 478.11 - Meaning of terms.

Code of Federal Regulations, 2011 CFR

2011-04-01

... than one shot, without manual reloading, by a single function of the trigger. The term shall also... than an antique firearm. The term shall not include (a) any shotgun shot or pellet not designed for use as the single, complete projectile load for one shotgun hull or casing, nor (b) any unloaded, non...

27 CFR 479.11 - Meaning of terms.

Code of Federal Regulations, 2013 CFR

2013-04-01

.... Any other weapon. Any weapon or device capable of being concealed on the person from which a shot can...) missile having an explosive or incendiary charge of more than one-quarter ounce, (5) mine, or (6) similar... have a bore of more than one-half inch in diameter, except a shotgun or shotgun shell which the...

27 CFR 478.11 - Meaning of terms.

Code of Federal Regulations, 2010 CFR

2010-04-01

... than one shot, without manual reloading, by a single function of the trigger. The term shall also... than an antique firearm. The term shall not include (a) any shotgun shot or pellet not designed for use as the single, complete projectile load for one shotgun hull or casing, nor (b) any unloaded, non...

27 CFR 479.11 - Meaning of terms.

Code of Federal Regulations, 2010 CFR

2010-04-01

.... Any other weapon. Any weapon or device capable of being concealed on the person from which a shot can...) missile having an explosive or incendiary charge of more than one-quarter ounce, (5) mine, or (6) similar... have a bore of more than one-half inch in diameter, except a shotgun or shotgun shell which the...

27 CFR 479.11 - Meaning of terms.

Code of Federal Regulations, 2012 CFR

2012-04-01

.... Any other weapon. Any weapon or device capable of being concealed on the person from which a shot can...) missile having an explosive or incendiary charge of more than one-quarter ounce, (5) mine, or (6) similar... have a bore of more than one-half inch in diameter, except a shotgun or shotgun shell which the...

27 CFR 478.11 - Meaning of terms.

Code of Federal Regulations, 2014 CFR

2014-04-01

... than one shot, without manual reloading, by a single function of the trigger. The term shall also... than an antique firearm. The term shall not include (a) any shotgun shot or pellet not designed for use as the single, complete projectile load for one shotgun hull or casing, nor (b) any unloaded, non...

27 CFR 478.11 - Meaning of terms.

Code of Federal Regulations, 2013 CFR

2013-04-01

... than one shot, without manual reloading, by a single function of the trigger. The term shall also... than an antique firearm. The term shall not include (a) any shotgun shot or pellet not designed for use as the single, complete projectile load for one shotgun hull or casing, nor (b) any unloaded, non...

27 CFR 479.11 - Meaning of terms.

Code of Federal Regulations, 2011 CFR

2011-04-01

.... Any other weapon. Any weapon or device capable of being concealed on the person from which a shot can...) missile having an explosive or incendiary charge of more than one-quarter ounce, (5) mine, or (6) similar... have a bore of more than one-half inch in diameter, except a shotgun or shotgun shell which the...

Comparative shotgun proteomic analysis of wild and domesticated Opuntia spp. species shows a metabolic adaptation through domestication.

PubMed

Pichereaux, Carole; Hernández-Domínguez, Eric E; Santos-Diaz, Maria Del Socorro; Reyes-Agüero, Antonio; Astello-García, Marizel; Guéraud, Françoise; Negre-Salvayre, Anne; Schiltz, Odile; Rossignol, Michel; Barba de la Rosa, Ana Paulina

2016-06-30

The Opuntia genus is widely distributed in America, but the highest richness of wild species are found in Mexico, as well as the most domesticated Opuntia ficus-indica, which is the most domesticated species and an important crop in agricultural economies of arid and semiarid areas worldwide. During domestication process, the Opuntia morphological characteristics were favored, such as less and smaller spines in cladodes and less seeds in fruits, but changes at molecular level are almost unknown. To obtain more insights about the Opuntia molecular changes through domestication, a shotgun proteomic analysis and database-dependent searches by homology was carried out. >1000 protein species were identified and by using a label-free quantitation method, the Opuntia proteomes were compared in order to identify differentially accumulated proteins among wild and domesticated species. Most of the changes were observed in glucose, secondary, and 1C metabolism, which correlate with the observed protein, fiber and phenolic compounds accumulation in Opuntia cladodes. Regulatory proteins, ribosomal proteins, and proteins related with response to stress were also observed in differential accumulation. These results provide new valuable data that will help to the understanding of the molecular changes of Opuntia species through domestication. Opuntia species are well adapted to dry and warm conditions in arid and semiarid regions worldwide, and they are highly productive plants showing considerable promises as an alternative food source. However, there is a gap regarding Opuntia molecular mechanisms that enable them to grow in extreme environmental conditions and how the domestication processes has changed them. In the present study, a shotgun analysis was carried out to characterize the proteomes of five Opuntia species selected by its domestication degree. Our results will help to a better understanding of proteomic features underlying the selection and specialization under evolution and domestication of Opuntia and will provide a platform for basic biology research and gene discovery. Copyright © 2016 Elsevier B.V. All rights reserved.

Unexpected Dominance of Elusive Acidobacteria in Early Industrial Soft Coal Slags

PubMed Central

Wegner, Carl-Eric; Liesack, Werner

2017-01-01

Acid mine drainage (AMD) and mine tailing environments are well-characterized ecosystems known to be dominated by organisms involved in iron- and sulfur-cycling. Here we examined the microbiology of industrial soft coal slags that originate from alum leaching, an ecosystem distantly related to AMD environments. Our study involved geochemical analyses, bacterial community profiling, and shotgun metagenomics. The slags still contained high amounts of alum constituents (aluminum, sulfur), which mediated direct and indirect effects on bacterial community structure. Bacterial groups typically found in AMD systems and mine tailings were not present. Instead, the soft coal slags were dominated by uncharacterized groups of Acidobacteria (DA052 [subdivision 2], KF-JG30-18 [subdivision 13]), Actinobacteria (TM214), Alphaproteobacteria (DA111), and Chloroflexi (JG37-AG-4), which have previously been detected primarily in peatlands and uranium waste piles. Shotgun metagenomics allowed us to reconstruct 13 high-quality Acidobacteria draft genomes, of which two genomes could be directly linked to dominating groups (DA052, KF-JG30-18) by recovered 16S rRNA gene sequences. Comparative genomics revealed broad carbon utilization capabilities for these two groups of elusive Acidobacteria, including polysaccharide breakdown (cellulose, xylan) and the competence to metabolize C1 compounds (ribulose monophosphate pathway) and lignin derivatives (dye-decolorizing peroxidases). Equipped with a broad range of efflux systems for metal cations and xenobiotics, DA052 and KF-JG30-18 may have a competitive advantage over other bacterial groups in this unique habitat. PMID:28642744

Label-free Quantitative Protein Profiling of vastus lateralis Muscle During Human Aging*

PubMed Central

Théron, Laëtitia; Gueugneau, Marine; Coudy, Cécile; Viala, Didier; Bijlsma, Astrid; Butler-Browne, Gillian; Maier, Andrea; Béchet, Daniel; Chambon, Christophe

2014-01-01

Sarcopenia corresponds to the loss of muscle mass occurring during aging, and is associated with a loss of muscle functionality. Proteomic links the muscle functional changes with protein expression pattern. To better understand the mechanisms involved in muscle aging, we performed a proteomic analysis of Vastus lateralis muscle in mature and older women. For this, a shotgun proteomic method was applied to identify soluble proteins in muscle, using a combination of high performance liquid chromatography and mass spectrometry. A label-free protein profiling was then conducted to quantify proteins and compare profiles from mature and older women. This analysis showed that 35 of the 366 identified proteins were linked to aging in muscle. Most of the proteins were under-represented in older compared with mature women. We built a functional interaction network linking the proteins differentially expressed between mature and older women. The results revealed that the main differences between mature and older women were defined by proteins involved in energy metabolism and proteins from the myofilament and cytoskeleton. This is the first time that label-free quantitative proteomics has been applied to study of aging mechanisms in human skeletal muscle. This approach highlights new elements for elucidating the alterations observed during aging and may lead to novel sarcopenia biomarkers. PMID:24217021

Label-free quantitative protein profiling of vastus lateralis muscle during human aging.

PubMed

Théron, Laëtitia; Gueugneau, Marine; Coudy, Cécile; Viala, Didier; Bijlsma, Astrid; Butler-Browne, Gillian; Maier, Andrea; Béchet, Daniel; Chambon, Christophe

2014-01-01

Sarcopenia corresponds to the loss of muscle mass occurring during aging, and is associated with a loss of muscle functionality. Proteomic links the muscle functional changes with protein expression pattern. To better understand the mechanisms involved in muscle aging, we performed a proteomic analysis of Vastus lateralis muscle in mature and older women. For this, a shotgun proteomic method was applied to identify soluble proteins in muscle, using a combination of high performance liquid chromatography and mass spectrometry. A label-free protein profiling was then conducted to quantify proteins and compare profiles from mature and older women. This analysis showed that 35 of the 366 identified proteins were linked to aging in muscle. Most of the proteins were under-represented in older compared with mature women. We built a functional interaction network linking the proteins differentially expressed between mature and older women. The results revealed that the main differences between mature and older women were defined by proteins involved in energy metabolism and proteins from the myofilament and cytoskeleton. This is the first time that label-free quantitative proteomics has been applied to study of aging mechanisms in human skeletal muscle. This approach highlights new elements for elucidating the alterations observed during aging and may lead to novel sarcopenia biomarkers.

Profiling of proteins secreted in the bovine oviduct reveals diverse functions of this luminal microenvironment.

PubMed

Pillai, Viju Vijayan; Weber, Darren M; Phinney, Brett S; Selvaraj, Vimal

2017-01-01

The oviductal microenvironment is a site for key events that involve gamete maturation, fertilization and early embryo development. Secretions into the oviductal lumen by either the lining epithelium or by transudation of plasma constituents are known to contain elements conducive for reproductive success. Although previous studies have identified some of these factors involved in reproduction, knowledge of secreted proteins in the oviductal fluid remains rudimentary with limited definition of function even in extensively studied species like cattle. In this study, we used a shotgun proteomics approach followed by bioinformatics sequence prediction to identify secreted proteins present in the bovine oviductal fluid (ex vivo) and secretions from the bovine oviductal epithelial cells (in vitro). From a total of 2087 proteins identified, 266 proteins could be classified as secreted, 109 (41%) of which were common for both in vivo and in vitro conditions. Pathway analysis indicated different classes of proteins that included growth factors, metabolic regulators, immune modulators, enzymes, and extracellular matrix components. Functional analysis revealed mechanisms in the oviductal lumen linked to immune homeostasis, gamete maturation, fertilization and early embryo development. These results point to several novel components that work together with known elements mediating functional homeostasis, and highlight the diversity of machinery associated with oviductal physiology and early events in cattle fertility.

A proteomic analysis of the chromoplasts isolated from sweet orange fruits [Citrus sinensis (L.) Osbeck].

PubMed

Zeng, Yunliu; Pan, Zhiyong; Ding, Yuduan; Zhu, Andan; Cao, Hongbo; Xu, Qiang; Deng, Xiuxin

2011-11-01

Here, a comprehensive proteomic analysis of the chromoplasts purified from sweet orange using Nycodenz density gradient centrifugation is reported. A GeLC-MS/MS shotgun approach was used to identify the proteins of pooled chromoplast samples. A total of 493 proteins were identified from purified chromoplasts, of which 418 are putative plastid proteins based on in silico sequence homology and functional analyses. Based on the predicted functions of these identified plastid proteins, a large proportion (∼60%) of the chromoplast proteome of sweet orange is constituted by proteins involved in carbohydrate metabolism, amino acid/protein synthesis, and secondary metabolism. Of note, HDS (hydroxymethylbutenyl 4-diphosphate synthase), PAP (plastid-lipid-associated protein), and psHSPs (plastid small heat shock proteins) involved in the synthesis or storage of carotenoid and stress response are among the most abundant proteins identified. A comparison of chromoplast proteomes between sweet orange and tomato suggested a high level of conservation in a broad range of metabolic pathways. However, the citrus chromoplast was characterized by more extensive carotenoid synthesis, extensive amino acid synthesis without nitrogen assimilation, and evidence for lipid metabolism concerning jasmonic acid synthesis. In conclusion, this study provides an insight into the major metabolic pathways as well as some unique characteristics of the sweet orange chromoplasts at the whole proteome level.

50 CFR 32.44 - Missouri.

Code of Federal Regulations, 2011 CFR

2011-10-01

... approved nontoxic shot (see § 32.2(k)). 2. We allow upland game hunting on the 131-acre mainland unit of... Statewide seasons using archery methods or shotguns using shot no larger than BB. 3. We allow upland game hunting on the Cora Island Unit only to shotguns with shot no larger than BB. C. Big Game Hunting. We...

Metagenomic Analyses Reveal the Involvement of Syntrophic Consortia in Methanol/Electricity Conversion in Microbial Fuel Cells

PubMed Central

Yamamuro, Ayaka; Kouzuma, Atsushi; Abe, Takashi; Watanabe, Kazuya

2014-01-01

Methanol is widely used in industrial processes, and as such, is discharged in large quantities in wastewater. Microbial fuel cells (MFCs) have the potential to recover electric energy from organic pollutants in wastewater; however, the use of MFCs to generate electricity from methanol has not been reported. In the present study, we developed single-chamber MFCs that generated electricity from methanol at the maximum power density of 220 mW m−2 (based on the projected area of the anode). In order to reveal how microbes generate electricity from methanol, pyrosequencing of 16S rRNA-gene amplicons and Illumina shotgun sequencing of metagenome were conducted. The pyrosequencing detected in abundance Dysgonomonas, Sporomusa, and Desulfovibrio in the electrolyte and anode and cathode biofilms, while Geobacter was detected only in the anode biofilm. Based on known physiological properties of these bacteria, it is considered that Sporomusa converts methanol into acetate, which is then utilized by Geobacter to generate electricity. This speculation is supported by results of shotgun metagenomics of the anode-biofilm microbes, which reconstructed relevant catabolic pathways in these bacteria. These results suggest that methanol is anaerobically catabolized by syntrophic bacterial consortia with electrodes as electron acceptors. PMID:24852573

Metagenomic analyses reveal the involvement of syntrophic consortia in methanol/electricity conversion in microbial fuel cells.

PubMed

Yamamuro, Ayaka; Kouzuma, Atsushi; Abe, Takashi; Watanabe, Kazuya

2014-01-01

Methanol is widely used in industrial processes, and as such, is discharged in large quantities in wastewater. Microbial fuel cells (MFCs) have the potential to recover electric energy from organic pollutants in wastewater; however, the use of MFCs to generate electricity from methanol has not been reported. In the present study, we developed single-chamber MFCs that generated electricity from methanol at the maximum power density of 220 mW m(-2) (based on the projected area of the anode). In order to reveal how microbes generate electricity from methanol, pyrosequencing of 16S rRNA-gene amplicons and Illumina shotgun sequencing of metagenome were conducted. The pyrosequencing detected in abundance Dysgonomonas, Sporomusa, and Desulfovibrio in the electrolyte and anode and cathode biofilms, while Geobacter was detected only in the anode biofilm. Based on known physiological properties of these bacteria, it is considered that Sporomusa converts methanol into acetate, which is then utilized by Geobacter to generate electricity. This speculation is supported by results of shotgun metagenomics of the anode-biofilm microbes, which reconstructed relevant catabolic pathways in these bacteria. These results suggest that methanol is anaerobically catabolized by syntrophic bacterial consortia with electrodes as electron acceptors.

Whole Genome Shotgun Analysis: the Resolving Power to Distinguish 2009 and 2010 Clinical Isolates of Salmonella Typhimurium Possessing the Same PFGE Type

USDA-ARS?s Scientific Manuscript database

Between 2009 and 2011, Salmonella enterica subsp. enterica serovar Typhimurium with the JPXX01.0014 pulse field gel electrophoresis (PFGE) type was isolated across the United States. The 2009 isolates were associated with an outbreak from an unidentified food source, while later isolates were linked...

Quantitation of isobaric phosphatidylcholine species in human plasma using a hybrid quadrupole linear ion trap mass spectrometer

USDA-ARS?s Scientific Manuscript database

Phosphatidylcholine (PC) species in human plasma are used as biomarkers of disease. PC biomarkers are often limited by the inability to separate isobaric PC. In this work, we developed a targeted shotgun approach for analysis of isobaric and isomeric PC. This approach is comprised of two mass spectr...

A high-throughput shotgun mutagenesis approach to mapping B-cell antibody epitopes.

PubMed

Davidson, Edgar; Doranz, Benjamin J

2014-09-01

Characterizing the binding sites of monoclonal antibodies (mAbs) on protein targets, their 'epitopes', can aid in the discovery and development of new therapeutics, diagnostics and vaccines. However, the speed of epitope mapping techniques has not kept pace with the increasingly large numbers of mAbs being isolated. Obtaining detailed epitope maps for functionally relevant antibodies can be challenging, particularly for conformational epitopes on structurally complex proteins. To enable rapid epitope mapping, we developed a high-throughput strategy, shotgun mutagenesis, that enables the identification of both linear and conformational epitopes in a fraction of the time required by conventional approaches. Shotgun mutagenesis epitope mapping is based on large-scale mutagenesis and rapid cellular testing of natively folded proteins. Hundreds of mutant plasmids are individually cloned, arrayed in 384-well microplates, expressed within human cells, and tested for mAb reactivity. Residues are identified as a component of a mAb epitope if their mutation (e.g. to alanine) does not support candidate mAb binding but does support that of other conformational mAbs or allows full protein function. Shotgun mutagenesis is particularly suited for studying structurally complex proteins because targets are expressed in their native form directly within human cells. Shotgun mutagenesis has been used to delineate hundreds of epitopes on a variety of proteins, including G protein-coupled receptor and viral envelope proteins. The epitopes mapped on dengue virus prM/E represent one of the largest collections of epitope information for any viral protein, and results are being used to design better vaccines and drugs. © 2014 John Wiley & Sons Ltd.

Effects of Fe and Mn deficiencies on the protein profiles of tomato (Solanum lycopersicum) xylem sap as revealed by shotgun analyses

USDA-ARS?s Scientific Manuscript database

The aim of this work was to study the effects of Fe and Mn deficiencies on the xylem sap proteome of tomato using a shotgun proteomic approach, with the final goal of elucidating plant response mechanisms to these stresses. This approach yielded 643 proteins reliably identified and quantified with 7...

Contemporary management of maxillofacial ballistic trauma.

PubMed

Breeze, J; Tong, D; Gibbons, A

2017-09-01

Ballistic maxillofacial trauma in the UK is fortunately relatively rare, and generally involves low velocity handguns and shotguns. Civilian terrorist events have, however, shown that all maxillofacial surgeons need to understand how to treat injuries from improvised explosive devices. Maxillofacial surgeons in the UK have also been responsible for the management of soldiers evacuated from Iraq and Afghanistan, and in this review we describe the newer types of treatment that have evolved from these conflicts, particularly that of damage-control maxillofacial surgery. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

Self-inflicted fatal shotgun wound from a homemade weapon.

PubMed

Tsuboi, Akio; Satoh, Fumiko; Seto, Yoshihisa; Osawa, Motoki

2014-03-01

A case of suicide, a male shot in the abdomen using a homemade weapon, is described herein. Postmortem examination revealed bleeding into the thoracic and abdominal cavities, a collapsed liver and other organs with numerous pellets. The contact-shot wound was not noticed during the initial investigation at the scene, but it was elucidated during autopsy. A simple launcher had been assembled from pipes of a clothes rack frame made for home use. The deceased had once owned a shotgun, but his firearm license had been relinquished 5 years prior. A shotgun shell that he had kept was used. Presumably, the injuries from the homemade weapon were not noticed initially because of strict gun control rules in Japan. Crown Copyright © 2013. Published by Elsevier Ireland Ltd. All rights reserved.

MerCat: a versatile k-mer counter and diversity estimator for database-independent property analysis obtained from metagenomic and/or metatranscriptomic sequencing data

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

White, Richard A.; Panyala, Ajay R.; Glass, Kevin A.

MerCat is a parallel, highly scalable and modular property software package for robust analysis of features in next-generation sequencing data. MerCat inputs include assembled contigs and raw sequence reads from any platform resulting in feature abundance counts tables. MerCat allows for direct analysis of data properties without reference sequence database dependency commonly used by search tools such as BLAST and/or DIAMOND for compositional analysis of whole community shotgun sequencing (e.g. metagenomes and metatranscriptomes).

Metagenomic Analysis of a Biphenyl-Degrading Soil Bacterial Consortium Reveals the Metabolic Roles of Specific Populations

PubMed Central

Garrido-Sanz, Daniel; Manzano, Javier; Martín, Marta; Redondo-Nieto, Miguel; Rivilla, Rafael

2018-01-01

Polychlorinated biphenyls (PCBs) are widespread persistent pollutants that cause several adverse health effects. Aerobic bioremediation of PCBs involves the activity of either one bacterial species or a microbial consortium. Using multiple species will enhance the range of PCB congeners co-metabolized since different PCB-degrading microorganisms exhibit different substrate specificity. We have isolated a bacterial consortium by successive enrichment culture using biphenyl (analog of PCBs) as the sole carbon and energy source. This consortium is able to grow on biphenyl, benzoate, and protocatechuate. Whole-community DNA extracted from the consortium was used to analyze biodiversity by Illumina sequencing of a 16S rRNA gene amplicon library and to determine the metagenome by whole-genome shotgun Illumina sequencing. Biodiversity analysis shows that the consortium consists of 24 operational taxonomic units (≥97% identity). The consortium is dominated by strains belonging to the genus Pseudomonas, but also contains betaproteobacteria and Rhodococcus strains. whole-genome shotgun (WGS) analysis resulted in contigs containing 78.3 Mbp of sequenced DNA, representing around 65% of the expected DNA in the consortium. Bioinformatic analysis of this metagenome has identified the genes encoding the enzymes implicated in three pathways for the conversion of biphenyl to benzoate and five pathways from benzoate to tricarboxylic acid (TCA) cycle intermediates, allowing us to model the whole biodegradation network. By genus assignment of coding sequences, we have also been able to determine that the three biphenyl to benzoate pathways are carried out by Rhodococcus strains. In turn, strains belonging to Pseudomonas and Bordetella are the main responsible of three of the benzoate to TCA pathways while the benzoate conversion into TCA cycle intermediates via benzoyl-CoA and the catechol meta-cleavage pathways are carried out by beta proteobacteria belonging to genera such as Achromobacter and Variovorax. We have isolated a Rhodococcus strain WAY2 from the consortium which contains the genes encoding the three biphenyl to benzoate pathways indicating that this strain is responsible for all the biphenyl to benzoate transformations. The presented results show that metagenomic analysis of consortia allows the identification of bacteria active in biodegradation processes and the assignment of specific reactions and pathways to specific bacterial groups. PMID:29497412

[Gunshot wounds caused by non-lethal ammunition on the porcine model post-mortem].

PubMed

Jabrocký, Peter; Pivko, Juraj; Vondráková, Mária; Tažký, Boris

2013-10-01

In this article we focus on the effects of so called non-lethal ammunition. We studied possible mechanism of firearm injury formation as a consequence of using firearm on the body, to present a more comprehensive material in wound ballistics. We pointed out possible actions of a projectile causes on human, respectively other animal organisms, as well as to a manner in which an injury is caused by rifles or shotguns using non-lethal ammunition with rubber projectiles. In the experiment, we have focused on macroscopic analysis of the tissue penetrated by a rubber projectile fired from a long firearm and pump-action shotgun while focusing on the anatomical-morphological analysis of entry wounds to determine the effectiveness respectively, the wounding potential of the projectile. The results of the experiment based on the macroscopic analysis of entry wounds, cavities and exit wounds, show that when a rubber projectile penetrates the body it causes loss of the tissue (i.e. the minus effect) and mechanical disruption of the tissue similar to lethal projectile. Based on the measures and ballistic computations we concluded that in specific cases, like for example in a close range hit, a penetration of vital organs can cause serious or even lethal injuries.

Analysis of lipid experiments (ALEX): a software framework for analysis of high-resolution shotgun lipidomics data.

PubMed

Husen, Peter; Tarasov, Kirill; Katafiasz, Maciej; Sokol, Elena; Vogt, Johannes; Baumgart, Jan; Nitsch, Robert; Ekroos, Kim; Ejsing, Christer S

2013-01-01

Global lipidomics analysis across large sample sizes produces high-content datasets that require dedicated software tools supporting lipid identification and quantification, efficient data management and lipidome visualization. Here we present a novel software-based platform for streamlined data processing, management and visualization of shotgun lipidomics data acquired using high-resolution Orbitrap mass spectrometry. The platform features the ALEX framework designed for automated identification and export of lipid species intensity directly from proprietary mass spectral data files, and an auxiliary workflow using database exploration tools for integration of sample information, computation of lipid abundance and lipidome visualization. A key feature of the platform is the organization of lipidomics data in "database table format" which provides the user with an unsurpassed flexibility for rapid lipidome navigation using selected features within the dataset. To demonstrate the efficacy of the platform, we present a comparative neurolipidomics study of cerebellum, hippocampus and somatosensory barrel cortex (S1BF) from wild-type and knockout mice devoid of the putative lipid phosphate phosphatase PRG-1 (plasticity related gene-1). The presented framework is generic, extendable to processing and integration of other lipidomic data structures, can be interfaced with post-processing protocols supporting statistical testing and multivariate analysis, and can serve as an avenue for disseminating lipidomics data within the scientific community. The ALEX software is available at www.msLipidomics.info.

Analysis of the Biotechnological Potential of a Lentinus crinitus Isolate in the Light of Its Secretome.

PubMed

Cambri, Geison; de Sousa, Mirta Mittelstedt Leal; Fonseca, Davi de Miranda; Marchini, Fabricio; da Silveira, Joana Lea Meira; Paba, Jaime

2016-12-02

Analysis of fungal secretomes is a prospection tool for the discovery of new catalysts with biotechnological applications. Since enzyme secretion is strongly modulated by environmental factors, evaluation of growth conditions is of utmost importance to achieve optimal enzyme production. In this work, a nonsequenced wood-rotting fungus, Lentinus crinitus, was used for secretome analysis by enzymatic assays and a proteomics approach. Enzyme production was assessed after the fungus was cultured in seven different carbon sources and three nitrogen-containing compounds. The biomass yields and secreted protein arrays differed drastically among growing conditions. A mixture of secreted extracts derived from solid and liquid cultures was inspected by shotgun mass spectrometry and two-dimensional gel electrophoresis (2-DE) prior to analysis via LC-MS/MS. Proteins were identified using mass spectrometry (MS)-driven BLAST. The spectrum of secreted proteins comprised CAZymes, oxidase/reductases, proteases, and lipase/esterases. Although preseparation by 2-DE improved the number of identifications (162) compared with the shotgun approach (98 identifications), the two strategies revealed similar protein patterns. Culture media with reduced water content stimulated the expression of oxidases/reductases, while hydrolases were induced during submerged fermentation. The diversity of proteins observed within both the CAZyme and oxidoreductase groups revealed in this fungus a powerful arsenal of enzymes dedicated to the breakdown and consumption of lignocellulose.

A simulated metagenomic approach for bacterial serotyping using shotgun genome sequences coupled with O-Antigen gene cluster analysis

USDA-ARS?s Scientific Manuscript database

Background: Accurate determination of food-borne pathogen serotype and genotype information is important for disease surveillance and outbreak source tracking. E. coli serotype O157:H7 and non-O157 of Shiga toxin-producing E. coli (STEC) serogroups, including O26, O45, O103, O111, O121, O145 (top ...

Random whole metagenomic sequencing for forensic discrimination of soils.

PubMed

Khodakova, Anastasia S; Smith, Renee J; Burgoyne, Leigh; Abarno, Damien; Linacre, Adrian

2014-01-01

Here we assess the ability of random whole metagenomic sequencing approaches to discriminate between similar soils from two geographically distinct urban sites for application in forensic science. Repeat samples from two parklands in residential areas separated by approximately 3 km were collected and the DNA was extracted. Shotgun, whole genome amplification (WGA) and single arbitrarily primed DNA amplification (AP-PCR) based sequencing techniques were then used to generate soil metagenomic profiles. Full and subsampled metagenomic datasets were then annotated against M5NR/M5RNA (taxonomic classification) and SEED Subsystems (metabolic classification) databases. Further comparative analyses were performed using a number of statistical tools including: hierarchical agglomerative clustering (CLUSTER); similarity profile analysis (SIMPROF); non-metric multidimensional scaling (NMDS); and canonical analysis of principal coordinates (CAP) at all major levels of taxonomic and metabolic classification. Our data showed that shotgun and WGA-based approaches generated highly similar metagenomic profiles for the soil samples such that the soil samples could not be distinguished accurately. An AP-PCR based approach was shown to be successful at obtaining reproducible site-specific metagenomic DNA profiles, which in turn were employed for successful discrimination of visually similar soil samples collected from two different locations.

Proteomic analysis and food-grade enzymes of Moringa oleifer Lam. a Lam. flower.

PubMed

Shi, Yanan; Wang, Xuefeng; Huang, Aixiang

2018-08-01

Moringa oleifer Lam. flower contain high-proteins and function nutrients. Many advances have been made to it, but there is still no proteomic information of this species. Total protein from the flowers applied shotgun 2DLC-MS/MS proteomic identified 9443 peptides corresponding to 4004 high-confidence proteins by Proteome Discoverer™ Software 2.1. These proteins were mostly distributed ranging between 40 and 70 kDa. Gene Ontology (GO) analysis indicated that the largest of the proteins were cytoplasm 72.7%, catalytic activity 61.5% and macromolecule metabolism 43.7%, and KEGG analysis revealed that the largest group of 129 proteins was involved in Ribosome to directing protein synthesis (translation). Moreover, a number of commercially important food-grade enzymes were commented, 261 proteins were annotated as carbohydrate-active enzymes, 16 protease, 22 proteins are assigned to the citrate cycle, which the top proteins were assigned to GH family, cysteine synthase and serine/threonine-protein phosphatase. These enzymes indicated that is a new source with potential use for fermentation and brewing industry, fruit and vegetable storage and the development of function peptides. Copyright © 2018 Elsevier B.V. All rights reserved.

Extracellular protein analysis of activated sludge and their functions in wastewater treatment plant by shotgun proteomics.

PubMed

Zhang, Peng; Shen, Yu; Guo, Jin-Song; Li, Chun; Wang, Han; Chen, You-Peng; Yan, Peng; Yang, Ji-Xiang; Fang, Fang

2015-07-10

In this work, proteins in extracellular polymeric substances extracted from anaerobic, anoxic and aerobic sludges of wastewater treatment plant (WWTP) were analyzed to probe their origins and functions. Extracellular proteins in WWTP sludges were identified using shotgun proteomics, and 130, 108 and 114 proteins in anaerobic, anoxic and aerobic samples were classified, respectively. Most proteins originated from cell and cell part, and their most major molecular functions were catalytic activity and binding activity. The results exhibited that the main roles of extracellular proteins in activated sludges were multivalence cations and organic molecules binding, as well as in catalysis and degradation. The catalytic activity proteins were more widespread in anaerobic sludge compared with those in anoxic and aerobic sludges. The structure difference between anaerobic and aerobic sludges could be associated with their catalytic activities proteins. The results also put forward a relation between the macro characteristics of activated sludges and micro functions of extracellular proteins in biological wastewater treatment process.

Protein and gene model inference based on statistical modeling in k-partite graphs.

PubMed

Gerster, Sarah; Qeli, Ermir; Ahrens, Christian H; Bühlmann, Peter

2010-07-06

One of the major goals of proteomics is the comprehensive and accurate description of a proteome. Shotgun proteomics, the method of choice for the analysis of complex protein mixtures, requires that experimentally observed peptides are mapped back to the proteins they were derived from. This process is also known as protein inference. We present Markovian Inference of Proteins and Gene Models (MIPGEM), a statistical model based on clearly stated assumptions to address the problem of protein and gene model inference for shotgun proteomics data. In particular, we are dealing with dependencies among peptides and proteins using a Markovian assumption on k-partite graphs. We are also addressing the problems of shared peptides and ambiguous proteins by scoring the encoding gene models. Empirical results on two control datasets with synthetic mixtures of proteins and on complex protein samples of Saccharomyces cerevisiae, Drosophila melanogaster, and Arabidopsis thaliana suggest that the results with MIPGEM are competitive with existing tools for protein inference.

SUPER-FOCUS: A tool for agile functional analysis of shotgun metagenomic data

DOE PAGES

Silva, Genivaldo Gueiros Z.; Green, Kevin T.; Dutilh, Bas E.; ...

2015-10-09

Analyzing the functional profile of a microbial community from unannotated shotgun sequencing reads is one of the important goals in metagenomics. Functional profiling has valuable applications in biological research because it identifies the abundances of the functional genes of the organisms present in the original sample, answering the question what they can do. Currently, available tools do not scale well with increasing data volumes, which is important because both the number and lengths of the reads produced by sequencing platforms keep increasing. Here, we introduce SUPER-FOCUS, SUbsystems Profile by databasE Reduction using FOCUS, an agile homology-based approach using a reducedmore » reference database to report the subsystems present in metagenomic datasets and profile their abundances. We tested SUPER-FOCUS with over 70 real metagenomes, the results showing that it accurately predicts the subsystems present in the profiled microbial communities, and is up to 1000 times faster than other tools.« less

Two cases of exenteration of the brain from Brenneke shotgun slugs.

PubMed

Karger, B; Banaschak, S

1997-01-01

A case of extended suicide resulted in two fatalities due to craniocerebral gunshots from a 12-gauge shotgun firing Brenneke shotgun slugs. In each case, the gunshot shattered the skull and the brain and in one case, large parts of the brain including a complete hemisphere were ejected similar to a "Krönlein shot". The location of the trajectory close to the base of the skull, the muzzle gases and the ballistic characteristics of the missile contributed to this rare form of head injury. The high mass and the large diameter of the lead missile do not necessitate a high muzzle velocity to crush large amounts of tissue or to produce an explosive type of head injury. The wadding material and the metal screw attached to the Brenneke slug can be of forensic significance.

Characterization of the human submandibular/sublingual saliva glycoproteome using lectin affinity chromatography coupled to Multidimensional Protein Identification Technology

PubMed Central

Gonzalez-Begne, Mireya; Lu, Bingwen; Liao, Lujian; Xu, Tao; Bedi, Gurrinder; Melvin, James E.; Yates, John R.

2011-01-01

In-depth analysis of the salivary proteome is fundamental to understanding the functions of salivary proteins in the oral cavity and to reveal disease biomarkers involved in different pathophysiological conditions, with the ultimate goal of improving patient diagnosis and prognosis. Submandibular and sublingual glands contribute saliva rich in glycoproteins to the total saliva output, making them valuable sources for glycoproteomic analysis. Lectin-affinity chromatography coupled to mass spectrometry-based shotgun proteomics was used to explore the submandibular/sublingual (SM/SL) saliva glycoproteome. A total of 262 N- and O-linked glycoproteins were identified by multidimensional protein identification technology (MudPIT). Only 38 were previously described in SM and SL salivas from the human salivary N-linked glycoproteome, while 224 were unique. Further comparison analysis with SM/SL saliva of the human saliva proteome, revealed 125 glycoproteins not formerly reported in this secretion. KEGG pathway analyses demonstrated that many of these glycoproteins are involved in processes such as complement and coagulation cascades, cell communication, glycosphingolipid biosynthesis neo-lactoseries, O-glycan biosynthesis, glycan structures-biosynthesis 2, starch and sucrose metabolism, peptidoglycan biosynthesis or others pathways. In summary, lectin-affinity chromatography coupled to MudPIT mass spectrometry identified many novel glycoproteins in SM/SL saliva. These new additions to the salivary proteome may prove to be a critical step for providing reliable biomarkers in the diagnosis of a myriad of oral and systemic diseases. PMID:21936497

Uncovering Trophic Interactions in Arthropod Predators through DNA Shotgun-Sequencing of Gut Contents

PubMed Central

Paula, Débora P.; Linard, Benjamin; Crampton-Platt, Alex; Srivathsan, Amrita; Timmermans, Martijn J. T. N.; Sujii, Edison R.; Pires, Carmen S. S.; Souza, Lucas M.; Andow, David A.; Vogler, Alfried P.

2016-01-01

Characterizing trophic networks is fundamental to many questions in ecology, but this typically requires painstaking efforts, especially to identify the diet of small generalist predators. Several attempts have been devoted to develop suitable molecular tools to determine predatory trophic interactions through gut content analysis, and the challenge has been to achieve simultaneously high taxonomic breadth and resolution. General and practical methods are still needed, preferably independent of PCR amplification of barcodes, to recover a broader range of interactions. Here we applied shotgun-sequencing of the DNA from arthropod predator gut contents, extracted from four common coccinellid and dermapteran predators co-occurring in an agroecosystem in Brazil. By matching unassembled reads against six DNA reference databases obtained from public databases and newly assembled mitogenomes, and filtering for high overlap length and identity, we identified prey and other foreign DNA in the predator guts. Good taxonomic breadth and resolution was achieved (93% of prey identified to species or genus), but with low recovery of matching reads. Two to nine trophic interactions were found for these predators, some of which were only inferred by the presence of parasitoids and components of the microbiome known to be associated with aphid prey. Intraguild predation was also found, including among closely related ladybird species. Uncertainty arises from the lack of comprehensive reference databases and reliance on low numbers of matching reads accentuating the risk of false positives. We discuss caveats and some future prospects that could improve the use of direct DNA shotgun-sequencing to characterize arthropod trophic networks. PMID:27622637

Soup to Tree: The Phylogeny of Beetles Inferred by Mitochondrial Metagenomics of a Bornean Rainforest Sample

PubMed Central

Crampton-Platt, Alex; Timmermans, Martijn J.T.N.; Gimmel, Matthew L.; Kutty, Sujatha Narayanan; Cockerill, Timothy D.; Vun Khen, Chey; Vogler, Alfried P.

2015-01-01

In spite of the growth of molecular ecology, systematics and next-generation sequencing, the discovery and analysis of diversity is not currently integrated with building the tree-of-life. Tropical arthropod ecologists are well placed to accelerate this process if all specimens obtained through mass-trapping, many of which will be new species, could be incorporated routinely into phylogeny reconstruction. Here we test a shotgun sequencing approach, whereby mitochondrial genomes are assembled from complex ecological mixtures through mitochondrial metagenomics, and demonstrate how the approach overcomes many of the taxonomic impediments to the study of biodiversity. DNA from approximately 500 beetle specimens, originating from a single rainforest canopy fogging sample from Borneo, was pooled and shotgun sequenced, followed by de novo assembly of complete and partial mitogenomes for 175 species. The phylogenetic tree obtained from this local sample was highly similar to that from existing mitogenomes selected for global coverage of major lineages of Coleoptera. When all sequences were combined only minor topological changes were induced against this reference set, indicating an increasingly stable estimate of coleopteran phylogeny, while the ecological sample expanded the tip-level representation of several lineages. Robust trees generated from ecological samples now enable an evolutionary framework for ecology. Meanwhile, the inclusion of uncharacterized samples in the tree-of-life rapidly expands taxon and biogeographic representation of lineages without morphological identification. Mitogenomes from shotgun sequencing of unsorted environmental samples and their associated metadata, placed robustly into the phylogenetic tree, constitute novel DNA “superbarcodes” for testing hypotheses regarding global patterns of diversity. PMID:25957318

Shedding genomic light on Aristotle's lantern.

PubMed

Sodergren, Erica; Shen, Yufeng; Song, Xingzhi; Zhang, Lan; Gibbs, Richard A; Weinstock, George M

2006-12-01

Sea urchins have proved fascinating to biologists since the time of Aristotle who compared the appearance of their bony mouth structure to a lantern in The History of Animals. Throughout modern times it has been a model system for research in developmental biology. Now, the genome of the sea urchin Strongylocentrotus purpuratus is the first echinoderm genome to be sequenced. A high quality draft sequence assembly was produced using the Atlas assembler to combine whole genome shotgun sequences with sequences from a collection of BACs selected to form a minimal tiling path along the genome. A formidable challenge was presented by the high degree of heterozygosity between the two haplotypes of the selected male representative of this marine organism. This was overcome by use of the BAC tiling path backbone, in which each BAC represents a single haplotype, as well as by improvements in the Atlas software. Another innovation introduced in this project was the sequencing of pools of tiling path BACs rather than individual BAC sequencing. The Clone-Array Pooled Shotgun Strategy greatly reduced the cost and time devoted to preparing shotgun libraries from BAC clones. The genome sequence was analyzed with several gene prediction methods to produce a comprehensive gene list that was then manually refined and annotated by a volunteer team of sea urchin experts. This latter annotation community edited over 9000 gene models and uncovered many unexpected aspects of the sea urchin genetic content impacting transcriptional regulation, immunology, sensory perception, and an organism's development. Analysis of the basic deuterostome genetic complement supports the sea urchin's role as a model system for deuterostome and, by extension, chordate development.

Human Papillomavirus Community in Healthy Persons, Defined by Metagenomics Analysis of Human Microbiome Project Shotgun Sequencing Data Sets

PubMed Central

Ma, Yingfei; Madupu, Ramana; Karaoz, Ulas; Nossa, Carlos W.; Yang, Liying; Yooseph, Shibu; Yachimski, Patrick S.; Brodie, Eoin L.; Nelson, Karen E.

2014-01-01

ABSTRACT Human papillomavirus (HPV) causes a number of neoplastic diseases in humans. Here, we show a complex normal HPV community in a cohort of 103 healthy human subjects, by metagenomics analysis of the shotgun sequencing data generated from the NIH Human Microbiome Project. The overall HPV prevalence was 68.9% and was highest in the skin (61.3%), followed by the vagina (41.5%), mouth (30%), and gut (17.3%). Of the 109 HPV types as well as additional unclassified types detected, most were undetectable by the widely used commercial kits targeting the vaginal/cervical HPV types. These HPVs likely represent true HPV infections rather than transitory exposure because of strong organ tropism and persistence of the same HPV types in repeat samples. Coexistence of multiple HPV types was found in 48.1% of the HPV-positive samples. Networking between HPV types, cooccurrence or exclusion, was detected in vaginal and skin samples. Large contigs assembled from short HPV reads were obtained from several samples, confirming their genuine HPV origin. This first large-scale survey of HPV using a shotgun sequencing approach yielded a comprehensive map of HPV infections among different body sites of healthy human subjects. IMPORTANCE This nonbiased survey indicates that the HPV community in healthy humans is much more complex than previously defined by widely used kits that are target selective for only a few high- and low-risk HPV types for cervical cancer. The importance of nononcogenic viruses in a mixed HPV infection could be for stimulating or inhibiting a coexisting oncogenic virus via viral interference or immune cross-reaction. Knowledge gained from this study will be helpful to guide the designing of epidemiological and clinical studies in the future to determine the impact of nononcogenic HPV types on the outcome of HPV infections. PMID:24522917

Fiber supplementation influences phylogenetic structure and functional capacity of the human intestinal microbiome: follow-up of a randomized controlled trial.

PubMed

Holscher, Hannah D; Caporaso, J Gregory; Hooda, Seema; Brulc, Jennifer M; Fahey, George C; Swanson, Kelly S

2015-01-01

In our published randomized, double-blind, placebo-controlled, 3-period crossover trial, healthy adult men (n = 21) consumed bars containing no supplemental fiber (placebo; NFC), polydextrose (21 g/d), and soluble corn fiber (SCF; 21 g/d) for 21 d each. Fecal specimens were collected between days 16 and 21 for fermentative end-product analysis and 16S ribosomal RNA bacterial gene amplification for bacterial taxa identification. Fiber supplementation decreased fecal putrefaction compounds and shifted abundances of several bacterial taxa. The objective was to perform whole-genome shotgun 454 pyrosequencing on the same fecal specimens collected in that clinical trial to obtain comprehensive fecal bacterial genome sequencing coverage and explore the full range of bacterial genetic information in the fecal microbiome, thereby using a systematic approach to study the impact of dietary fiber supplementation on fecal metabolites, bacterial taxa, and bacterial metagenomes. Fecal samples were subjected to whole-genome shotgun 454 pyrosequencing to identify both fecal bacterial populations present and their functional genetic capacity. Whole-genome shotgun sequencing results revealed that fiber consumption shifted the Bacteroidetes:Firmicutes ratio, increasing the relative abundance of Bacteroidetes 12 ± 2% and 13 ± 2% with polydextrose and SCF, respectively, compared with NFC. Bivariate correlations showed a positive correlation between the Bacteroidetes:Firmicutes ratio and total dietary fiber intake but not body mass index. Principal coordinates analysis of Bray-Curtis distances indicated that bacterial gene composition was more similar in participants consuming fibers (polydextrose and SCF combined) in comparison with NFC. Shifts in bacterial gene abundances after polydextrose and SCF supplementation included genes associated with carbohydrate, amino acid, and lipid metabolism, as well as metabolism of cofactors and vitamins. This study conveys novel information about the impact of dietary fiber supplementation on the phylogenetic structure and functional capacity of the fecal microbiome of healthy adults. © 2015 American Society for Nutrition.

Astragaloside IV Attenuates Glutamate-Induced Neurotoxicity in PC12 Cells through Raf-MEK-ERK Pathway.

PubMed

Yue, Rongcai; Li, Xia; Chen, Bingyang; Zhao, Jing; He, Weiwei; Yuan, Hu; Yuan, Xing; Gao, Na; Wu, Guozhen; Jin, Huizi; Shan, Lei; Zhang, Weidong

2015-01-01

Astragaloside IV (AGS-IV) is a main active ingredient of Astragalus membranaceus Bunge, a medicinal herb prescribed as an immunostimulant, hepatoprotective, antiperspirant, a diuretic or a tonic as documented in Chinese Materia Medica. In the present study, we employed a high-throughput comparative proteomic approach based on 2D-nano-LC-MS/MS to investigate the possible mechanism of action involved in the neuroprotective effect of AGS-IV against glutamate-induced neurotoxicity in PC12 cells. Differential proteins were identified, among which 13 proteins survived the stringent filter criteria and were further included for functional discussion. Two proteins (vimentin and Gap43) were randomly selected, and their expression levels were further confirmed by western blots analysis. The results matched well with those of proteomics. Furthermore, network analysis of protein-protein interactions (PPI) and pathways enrichment with AGS-IV associated proteins were carried out to illustrate its underlying molecular mechanism. Proteins associated with signal transduction, immune system, signaling molecules and interaction, and energy metabolism play important roles in neuroprotective effect of AGS-IV and Raf-MEK-ERK pathway was involved in the neuroprotective effect of AGS-IV against glutamate-induced neurotoxicity in PC12 cells. This study demonstrates that comparative proteomics based on shotgun approach is a valuable tool for molecular mechanism studies, since it allows the simultaneously evaluate the global proteins alterations.

A proteomic analysis of the chromoplasts isolated from sweet orange fruits [Citrus sinensis (L.) Osbeck

PubMed Central

Zeng, Yunliu; Pan, Zhiyong; Ding, Yuduan; Zhu, Andan; Cao, Hongbo; Xu, Qiang; Deng, Xiuxin

2011-01-01

Here, a comprehensive proteomic analysis of the chromoplasts purified from sweet orange using Nycodenz density gradient centrifugation is reported. A GeLC-MS/MS shotgun approach was used to identify the proteins of pooled chromoplast samples. A total of 493 proteins were identified from purified chromoplasts, of which 418 are putative plastid proteins based on in silico sequence homology and functional analyses. Based on the predicted functions of these identified plastid proteins, a large proportion (∼60%) of the chromoplast proteome of sweet orange is constituted by proteins involved in carbohydrate metabolism, amino acid/protein synthesis, and secondary metabolism. Of note, HDS (hydroxymethylbutenyl 4-diphosphate synthase), PAP (plastid-lipid-associated protein), and psHSPs (plastid small heat shock proteins) involved in the synthesis or storage of carotenoid and stress response are among the most abundant proteins identified. A comparison of chromoplast proteomes between sweet orange and tomato suggested a high level of conservation in a broad range of metabolic pathways. However, the citrus chromoplast was characterized by more extensive carotenoid synthesis, extensive amino acid synthesis without nitrogen assimilation, and evidence for lipid metabolism concerning jasmonic acid synthesis. In conclusion, this study provides an insight into the major metabolic pathways as well as some unique characteristics of the sweet orange chromoplasts at the whole proteome level. PMID:21841170

Iatrogenic artefacts attributable to traditional cupping therapy in a shotgun fatality.

PubMed

Cavlak, Mehmet; Özkök, Alper; Sarı, Serhat; Dursun, Ahmet; Akar, Taner; Karapirli, Mustafa; Demirel, Birol

2015-10-01

Cupping is a traditional treatment method that has been used for thousands of years to diminish pain, restore appetite and improve digestion, remove tendency to faint or remove 'bad blood' from the body. The suction of the cup is created by fire or mechanical devices. This procedure may result in circular erythema, petechiae, purpura, ecchymosis, burns and may be mistaken for trauma-related ecchymosis or livor mortis. Forty-year-old male was died by shotgun injuries in the same day of the wounding. Circular ecchymoses were observed on the forehead, within the scalp of occipital region, the back of the neck, and on the back. They were defined as ecchymoses in the first examination made by a general practitioner. In the external examination during the legal autopsy superficial incisions were observed on the circular ecchymoses. The shape, localization and color of and the characteristics of incisions on the circular lesions were concluded to be caused by the dry cupping therapy and wet cupping therapy procedures. These lesions and their formation mechanisms should be well-known by the forensic medical examiners and the other medical personnel involved in the forensic medical examination. Copyright © 2015 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Complete Genome Sequences of Two Methicillin-Resistant Staphylococcus haemolyticus Isolates of Multilocus Sequence Type 25, First Detected by Shotgun Metagenomics.

PubMed

Couto, Natacha; Chlebowicz, Monika A; Raangs, Erwin C; Friedrich, Alex W; Rossen, John W

2018-04-05

The emergence of nosocomial infections by multidrug-resistant Staphylococcus haemolyticus isolates has been reported in several European countries. Here, we report the first two complete genome sequences of S. haemolyticus sequence type 25 (ST25) isolates 83131A and 83131B. Both isolates were isolated from the same clinical sample and were first identified through shotgun metagenomics. Copyright © 2018 Couto et al.

Blood from a turnip: tissue origin of low-coverage shotgun sequencing libraries affects recovery of mitogenome sequences

USGS Publications Warehouse

Barker, F. Keith; Oyler-McCance, Sara; Tomback, Diana F.

2015-01-01

Next generation sequencing methods allow rapid, economical accumulation of data that have many applications, even at relatively low levels of genome coverage. However, the utility of shotgun sequencing data sets for specific goals may vary depending on the biological nature of the samples sequenced. We show that the ability to assemble mitogenomes from three avian samples of two different tissue types varies widely. In particular, data with coverage typical of microsatellite development efforts (∼1×) from DNA extracted from avian blood failed to cover even 50% of the mitogenome, relative to at least 500-fold coverage from muscle-derived data. Researchers should consider possible applications of their data and select the tissue source for their work accordingly. Practitioners analyzing low-coverage shotgun sequencing data (including for microsatellite locus development) should consider the potential benefits of mitogenome assembly, including internal barcode verification of species identity, mitochondrial primer development, and phylogenetics.

MULTI-DIMENSIONAL MASS SPECTROMETRY-BASED SHOTGUN LIPIDOMICS AND NOVEL STRATEGIES FOR LIPIDOMIC ANALYSES

PubMed Central

Han, Xianlin; Yang, Kui; Gross, Richard W.

2011-01-01

Since our last comprehensive review on multi-dimensional mass spectrometry-based shotgun lipidomics (Mass Spectrom. Rev. 24 (2005), 367), many new developments in the field of lipidomics have occurred. These developments include new strategies and refinements for shotgun lipidomic approaches that use direct infusion, including novel fragmentation strategies, identification of multiple new informative dimensions for mass spectrometric interrogation, and the development of new bioinformatic approaches for enhanced identification and quantitation of the individual molecular constituents that comprise each cell’s lipidome. Concurrently, advances in liquid chromatography-based platforms and novel strategies for quantitative matrix-assisted laser desorption/ionization mass spectrometry for lipidomic analyses have been developed. Through the synergistic use of this repertoire of new mass spectrometric approaches, the power and scope of lipidomics has been greatly expanded to accelerate progress toward the comprehensive understanding of the pleiotropic roles of lipids in biological systems. PMID:21755525

Short term changes in the proteome of human cerebral organoids induced by 5-MeO-DMT.

PubMed

Dakic, Vanja; Minardi Nascimento, Juliana; Costa Sartore, Rafaela; Maciel, Renata de Moraes; de Araujo, Draulio B; Ribeiro, Sidarta; Martins-de-Souza, Daniel; Rehen, Stevens K

2017-10-09

Dimethyltryptamines are entheogenic serotonin-like molecules present in traditional Amerindian medicine recently associated with cognitive gains, antidepressant effects, and changes in brain areas related to attention. Legal restrictions and the lack of adequate experimental models have limited the understanding of how such substances impact human brain metabolism. Here we used shotgun mass spectrometry to explore proteomic differences induced by 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) on human cerebral organoids. Out of the 6,728 identified proteins, 934 were found differentially expressed in 5-MeO-DMT-treated cerebral organoids. In silico analysis reinforced previously reported anti-inflammatory actions of 5-MeO-DMT and revealed modulatory effects on proteins associated with long-term potentiation, the formation of dendritic spines, including those involved in cellular protrusion formation, microtubule dynamics, and cytoskeletal reorganization. Our data offer the first insight about molecular alterations caused by 5-MeO-DMT in human cerebral organoids.

Vemurafenib resistance signature by proteome analysis offers new strategies and rational therapeutic concepts.

PubMed

Paulitschke, Verena; Berger, Walter; Paulitschke, Philipp; Hofstätter, Elisabeth; Knapp, Bernhard; Dingelmaier-Hovorka, Ruth; Födinger, Dagmar; Jäger, Walter; Szekeres, Thomas; Meshcheryakova, Anastasia; Bileck, Andrea; Pirker, Christine; Pehamberger, Hubert; Gerner, Christopher; Kunstfeld, Rainer

2015-03-01

The FDA-approved BRAF inhibitor vemurafenib achieves outstanding clinical response rates in patients with melanoma, but early resistance is common. Understanding the pathologic mechanisms of drug resistance and identification of effective therapeutic alternatives are key scientific challenges in the melanoma setting. Using proteomic techniques, including shotgun analysis and 2D-gel electrophoresis, we identified a comprehensive signature of the vemurafenib-resistant M24met in comparison with the vemurafenib-sensitive A375 melanoma cell line. The resistant cells were characterized by loss of differentiation, induction of transformation, enhanced expression of the lysosomal compartment, increased potential for metastasis, migration, adherence and Ca2(+) ion binding, enhanced expression of the MAPK pathway and extracellular matrix proteins, and epithelial-mesenchymal transformation. The main features were verified by shotgun analysis with QEXACTIVE orbitrap MS, electron microscopy, lysosomal staining, Western blotting, and adherence assay in a VM-1 melanoma cell line with acquired vemurafenib resistance. On the basis of the resistance profile, we were able to successfully predict that a novel resveratrol-derived COX-2 inhibitor, M8, would be active against the vemurafenib-resistant but not the vemurafenib-sensitive melanoma cells. Using high-throughput methods for cell line and drug characterization may thus offer a new way to identify key features of vemurafenib resistance, facilitating the design of effective rational therapeutic alternatives. ©2015 American Association for Cancer Research.

Alkylation Damage by Lipid Electrophiles Targets Functional Protein Systems*

PubMed Central

Codreanu, Simona G.; Ullery, Jody C.; Zhu, Jing; Tallman, Keri A.; Beavers, William N.; Porter, Ned A.; Marnett, Lawrence J.; Zhang, Bing; Liebler, Daniel C.

2014-01-01

Protein alkylation by reactive electrophiles contributes to chemical toxicities and oxidative stress, but the functional impact of alkylation damage across proteomes is poorly understood. We used Click chemistry and shotgun proteomics to profile the accumulation of proteome damage in human cells treated with lipid electrophile probes. Protein target profiles revealed three damage susceptibility classes, as well as proteins that were highly resistant to alkylation. Damage occurred selectively across functional protein interaction networks, with the most highly alkylation-susceptible proteins mapping to networks involved in cytoskeletal regulation. Proteins with lower damage susceptibility mapped to networks involved in protein synthesis and turnover and were alkylated only at electrophile concentrations that caused significant toxicity. Hierarchical susceptibility of proteome systems to alkylation may allow cells to survive sublethal damage while protecting critical cell functions. PMID:24429493

Identification of Antigenic Glycans from Schistosoma mansoni by Using a Shotgun Egg Glycan Microarray.

PubMed

Mickum, Megan L; Prasanphanich, Nina Salinger; Song, Xuezheng; Dorabawila, Nelum; Mandalasi, Msano; Lasanajak, Yi; Luyai, Anthony; Secor, W Evan; Wilkins, Patricia P; Van Die, Irma; Smith, David F; Nyame, A Kwame; Cummings, Richard D; Rivera-Marrero, Carlos A

2016-05-01

Infection of mammals by the parasitic helminth Schistosoma mansoni induces antibodies to glycan antigens in worms and eggs, but the differential nature of the immune response among infected mammals is poorly understood. To better define these responses, we used a shotgun glycomics approach in which N-glycans from schistosome egg glycoproteins were prepared, derivatized, separated, and used to generate an egg shotgun glycan microarray. This array was interrogated with sera from infected mice, rhesus monkeys, and humans and with glycan-binding proteins and antibodies to gather information about the structures of antigenic glycans, which also were analyzed by mass spectrometry. A major glycan antigen targeted by IgG from different infected species is the FLDNF epitope [Fucα3GalNAcβ4(Fucα3)GlcNAc-R], which is also recognized by the IgG monoclonal antibody F2D2. The FLDNF antigen is expressed by all life stages of the parasite in mammalian hosts, and F2D2 can kill schistosomula in vitro in a complement-dependent manner. Different antisera also recognized other glycan determinants, including core β-xylose and highly fucosylated glycans. Thus, the natural shotgun glycan microarray of schistosome eggs is useful in identifying antigenic glycans and in developing new anti-glycan reagents that may have diagnostic applications and contribute to developing new vaccines against schistosomiasis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Identification of Antigenic Glycans from Schistosoma mansoni by Using a Shotgun Egg Glycan Microarray

PubMed Central

Mickum, Megan L.; Prasanphanich, Nina Salinger; Song, Xuezheng; Dorabawila, Nelum; Mandalasi, Msano; Lasanajak, Yi; Luyai, Anthony; Secor, W. Evan; Wilkins, Patricia P.; Van Die, Irma; Smith, David F.; Nyame, A. Kwame

2016-01-01

Infection of mammals by the parasitic helminth Schistosoma mansoni induces antibodies to glycan antigens in worms and eggs, but the differential nature of the immune response among infected mammals is poorly understood. To better define these responses, we used a shotgun glycomics approach in which N-glycans from schistosome egg glycoproteins were prepared, derivatized, separated, and used to generate an egg shotgun glycan microarray. This array was interrogated with sera from infected mice, rhesus monkeys, and humans and with glycan-binding proteins and antibodies to gather information about the structures of antigenic glycans, which also were analyzed by mass spectrometry. A major glycan antigen targeted by IgG from different infected species is the FLDNF epitope [Fucα3GalNAcβ4(Fucα3)GlcNAc-R], which is also recognized by the IgG monoclonal antibody F2D2. The FLDNF antigen is expressed by all life stages of the parasite in mammalian hosts, and F2D2 can kill schistosomula in vitro in a complement-dependent manner. Different antisera also recognized other glycan determinants, including core β-xylose and highly fucosylated glycans. Thus, the natural shotgun glycan microarray of schistosome eggs is useful in identifying antigenic glycans and in developing new anti-glycan reagents that may have diagnostic applications and contribute to developing new vaccines against schistosomiasis. PMID:26883596

Improved False Discovery Rate Estimation Procedure for Shotgun Proteomics.

PubMed

Keich, Uri; Kertesz-Farkas, Attila; Noble, William Stafford

2015-08-07

Interpreting the potentially vast number of hypotheses generated by a shotgun proteomics experiment requires a valid and accurate procedure for assigning statistical confidence estimates to identified tandem mass spectra. Despite the crucial role such procedures play in most high-throughput proteomics experiments, the scientific literature has not reached a consensus about the best confidence estimation methodology. In this work, we evaluate, using theoretical and empirical analysis, four previously proposed protocols for estimating the false discovery rate (FDR) associated with a set of identified tandem mass spectra: two variants of the target-decoy competition protocol (TDC) of Elias and Gygi and two variants of the separate target-decoy search protocol of Käll et al. Our analysis reveals significant biases in the two separate target-decoy search protocols. Moreover, the one TDC protocol that provides an unbiased FDR estimate among the target PSMs does so at the cost of forfeiting a random subset of high-scoring spectrum identifications. We therefore propose the mix-max procedure to provide unbiased, accurate FDR estimates in the presence of well-calibrated scores. The method avoids biases associated with the two separate target-decoy search protocols and also avoids the propensity for target-decoy competition to discard a random subset of high-scoring target identifications.

Characterization of the stromatolite microbiome from Little Darby Island, The Bahamas using predictive and whole shotgun metagenomic analysis.

PubMed

Casaburi, Giorgio; Duscher, Alexandrea A; Reid, R Pamela; Foster, Jamie S

2016-05-01

Modern stromatolites represent ideal ecosystems to understand the biological processes required for the precipitation of carbonate due to their long evolutionary history and occurrence in a wide range of habitats. However, most of the prior molecular work on stromatolites has focused on understanding the taxonomic complexity and not fully elucidating the functional capabilities of these systems. Here, we begin to characterize the microbiome associated with stromatolites of Little Darby Island, The Bahamas using predictive metagenomics of the 16S rRNA gene coupled with direct whole shotgun sequencing. The metagenomic analysis of the Little Darby stromatolites revealed many shared taxa and core pathways associated with biologically induced carbonate precipitation, suggesting functional convergence within Bahamian stromatolites. A comparison of the Little Darby stromatolites with other lithifying microbial ecosystems also revealed that although factors, such as geographic location and salinity, do drive some differences within the population, there are extensive similarities within the microbial populations. These results suggest that for stromatolite formation, 'who' is in the community is not as critical as metabolic activities and environmental interactions. Together, these analyses help improve our understanding of the similarities among lithifying ecosystems and provide an important first step in characterizing the shared microbiome of modern stromatolites. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Improved False Discovery Rate Estimation Procedure for Shotgun Proteomics

PubMed Central

2016-01-01

Interpreting the potentially vast number of hypotheses generated by a shotgun proteomics experiment requires a valid and accurate procedure for assigning statistical confidence estimates to identified tandem mass spectra. Despite the crucial role such procedures play in most high-throughput proteomics experiments, the scientific literature has not reached a consensus about the best confidence estimation methodology. In this work, we evaluate, using theoretical and empirical analysis, four previously proposed protocols for estimating the false discovery rate (FDR) associated with a set of identified tandem mass spectra: two variants of the target-decoy competition protocol (TDC) of Elias and Gygi and two variants of the separate target-decoy search protocol of Käll et al. Our analysis reveals significant biases in the two separate target-decoy search protocols. Moreover, the one TDC protocol that provides an unbiased FDR estimate among the target PSMs does so at the cost of forfeiting a random subset of high-scoring spectrum identifications. We therefore propose the mix-max procedure to provide unbiased, accurate FDR estimates in the presence of well-calibrated scores. The method avoids biases associated with the two separate target-decoy search protocols and also avoids the propensity for target-decoy competition to discard a random subset of high-scoring target identifications. PMID:26152888

Characterization of the stromatolite microbiome from Little Darby Island, The Bahamas using predictive and whole shotgun metagenomic analysis

PubMed Central

Casaburi, Giorgio; Duscher, Alexandrea A.; Reid, R. Pamela; Foster, Jamie S.

2018-01-01

Summary Modern stromatolites represent ideal ecosystems to understand the biological processes required for the precipitation of carbonate due to their long evolutionary history and occurrence in a wide range of habitats. However, most of the prior molecular work on stromatolites has focused on understanding the taxonomic complexity and not fully elucidating the functional capabilities of these systems. Here, we begin to characterize the microbiome associated with stromatolites of Little Darby Island, The Bahamas using predictive metagenomics of the 16S rRNA gene coupled with direct whole shotgun sequencing. The metagenomic analysis of the Little Darby stromatolites revealed many shared taxa and core pathways associated with biologically induced carbonate precipitation, suggesting functional convergence within Bahamian stromatolites. A comparison of the Little Darby stromatolites with other lithifying microbial ecosystems also revealed that although factors, such as geographic location and salinity, do drive some differences within the population, there are extensive similarities within the microbial populations. These results suggest that for stromatolite formation, ‘who’ is in the community is not as critical as metabolic activities and environmental interactions. Together, these analyses help improve our understanding of the similarities among lithifying ecosystems and provide an important first step in characterizing the shared microbiome of modern stromatolites. PMID:26471001

Shotgun Pyrosequencing Metagenomic Analyses of Dusts from Swine Confinement and Grain Facilities

PubMed Central

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

2014-01-01

Inhalation of agricultural dusts causes inflammatory reactions and symptoms such as headache, fever, and malaise, which can progress to chronic airway inflammation and associated diseases, e.g. asthma, chronic bronchitis, chronic obstructive pulmonary disease, and hypersensitivity pneumonitis. Although in many agricultural environments feed particles are the major constituent of these dusts, the inflammatory responses that they provoke are likely attributable to particle-associated bacteria, archaebacteria, fungi, and viruses. In this study, we performed shotgun pyrosequencing metagenomic analyses of DNA from dusts from swine confinement facilities or grain elevators, with comparisons to dusts from pet-free households. DNA sequence alignment showed that 19% or 62% of shotgun pyrosequencing metagenomic DNA sequence reads from swine facility or household dusts, respectively, were of swine or human origin, respectively. In contrast only 2% of such reads from grain elevator dust were of mammalian origin. These metagenomic shotgun reads of mammalian origin were excluded from our analyses of agricultural dust microbiota. The ten most prevalent bacterial taxa identified in swine facility compared to grain elevator or household dust were comprised of 75%, 16%, and 42% gram-positive organisms, respectively. Four of the top five swine facility dust genera were assignable (Clostridium, Lactobacillus, Ruminococcus, and Eubacterium, ranging from 4% to 19% relative abundance). The relative abundances of these four genera were lower in dust from grain elevators or pet-free households. These analyses also highlighted the predominance in swine facility dust of Firmicutes (70%) at the phylum level, Clostridia (44%) at the Class level, and Clostridiales at the Order level (41%). In summary, shotgun pyrosequencing metagenomic analyses of agricultural dusts show that they differ qualitatively and quantitatively at the level of microbial taxa present, and that the bioinformatic analyses used for such studies must be carefully designed to avoid the potential contribution of non-microbial DNA, e.g. from resident mammals. PMID:24748147

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

PubMed

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

2014-01-01

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

A Novel Prosthetic Joint Infection Pathogen, Mycoplasma salivarium, Identified by Metagenomic Shotgun Sequencing.

PubMed

Thoendel, Matthew; Jeraldo, Patricio; Greenwood-Quaintance, Kerryl E; Chia, Nicholas; Abdel, Matthew P; Steckelberg, James M; Osmon, Douglas R; Patel, Robin

2017-07-15

Defining the microbial etiology of culture-negative prosthetic joint infection (PJI) can be challenging. Metagenomic shotgun sequencing is a new tool to identify organisms undetected by conventional methods. We present a case where metagenomics was used to identify Mycoplasma salivarium as a novel PJI pathogen in a patient with hypogammaglobulinemia. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Analysis of soybean tissue culture protein dynamics using difference gel electrophoresis.

PubMed

Miernyk, Ján A; Jett, Alissa A; Johnston, Mark L

2016-01-01

Excised hypocotyls from developing soybean (Glycine max (L.) merr. cv. Jack) were cultivated on agar-solidified medium until callus formed. The calli were then propagated in liquid medium until stable, relatively uniform, finely-divided suspension cultures were obtained. Cells were typically transferred to fresh medium at 7-day intervals. Cultures were harvested by filtration five days (early log phase) or eight days (late log phase) after transfer. In order to evaluate dynamic changes, both intracellular and extracellular proteins were analyzed by 2-dimensional difference gel electrophoresis. Selected spots were subjected to in-gel tryptic-digestion and the resultant peptides were analyzed by nLC-MS/MS. In follow-up studies gel-free shot-gun analyses led to identification of 367 intracellular proteins and 188 extracellular proteins. The significance of the described research is two-fold. First a gel-based proteomics method was applied to the study of the dynamics of the secretome (extracellular proteins). Second, results of a shot-gun non-gel based proteomic survey of both cellular and extracellular proteins are presented. Published by Elsevier B.V.

Pepitome: evaluating improved spectral library search for identification complementarity and quality assessment

PubMed Central

Dasari, Surendra; Chambers, Matthew C.; Martinez, Misti A.; Carpenter, Kristin L.; Ham, Amy-Joan L.; Vega-Montoto, Lorenzo J.; Tabb, David L.

2012-01-01

Spectral libraries have emerged as a viable alternative to protein sequence databases for peptide identification. These libraries contain previously detected peptide sequences and their corresponding tandem mass spectra (MS/MS). Search engines can then identify peptides by comparing experimental MS/MS scans to those in the library. Many of these algorithms employ the dot product score for measuring the quality of a spectrum-spectrum match (SSM). This scoring system does not offer a clear statistical interpretation and ignores fragment ion m/z discrepancies in the scoring. We developed a new spectral library search engine, Pepitome, which employs statistical systems for scoring SSMs. Pepitome outperformed the leading library search tool, SpectraST, when analyzing data sets acquired on three different mass spectrometry platforms. We characterized the reliability of spectral library searches by confirming shotgun proteomics identifications through RNA-Seq data. Applying spectral library and database searches on the same sample revealed their complementary nature. Pepitome identifications enabled the automation of quality analysis and quality control (QA/QC) for shotgun proteomics data acquisition pipelines. PMID:22217208

The current status and portability of our sequence handling software.

PubMed Central

Staden, R

1986-01-01

I describe the current status of our sequence analysis software. The package contains a comprehensive suite of programs for managing large shotgun sequencing projects, a program containing 61 functions for analysing single sequences and a program for comparing pairs of sequences for similarity. The programs that have been described before have been improved by the addition of new functions and by being made very much easier to use. The major interactive programs have 125 pages of online help available from within them. Several new programs are described including screen editing of aligned gel readings for shotgun sequencing projects; a method to highlight errors in aligned gel readings, new methods for searching for putative signals in sequences. We use the programs on a VAX computer but the whole package has been rewritten to make it easy to transport it to other machines. I believe the programs will now run on any machine with a FORTRAN77 compiler and sufficient memory. We are currently putting the programs onto an IBM PC XT/AT and another micro running under UNIX. PMID:3511446

Differences in shotgun protein expression profile between superficial bladder transitional cell carcinoma and normal urothelium.

PubMed

Niu, Hai Tao; Zhang, Yi Bing; Jiang, Hai Ping; Cheng, Bo; Sun, Guang; Wang, Yi; E, Ya Jun; Pang, De Quan; Chang, Ji Wu

2009-01-01

This study was undertaken to identify differences in protein expression profiles between superficial bladder transitional cell carcinoma (BTCC) and normal urothelial cells. We used laser capture microdissection (LCM) to harvest purified cells, and used two-dimensional liquid chromatography (2D-LC) followed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) to separate and identify the peptide mixture. A total of 440/438 proteins commonly appeared in 4 paired specimens. Multi-step bioinformatic procedures were used for the analysis of identified proteins; 175/179 of the 293/287 proteins that were specific expressed in tumor/normal cells own gene ontology (GO) biological process annotation. Compared with the entire list of the international protein index (IPI), there are 52/46 GO terms exhibited as enriched and 6/10 exhibited as depleted, respectively. Significantly altered pathways between tumor and normal cells mainly include oxidative phosphorylation, focal adhesion, etc. Finally, descriptive statistics show that the shotgun proteomics strategy has practice directive significance for biomarker discovery by two-dimensional electrophoresis (2-DE) technology.

Mass Defect Labeling of Cysteine for Improving Peptide Assignment in Shotgun Proteomic Analyses

PubMed Central

Hernandez, Hilda; Niehauser, Sarah; Boltz, Stacey A.; Gawandi, Vijay; Phillips, Robert S.; Amster, I. Jonathan

2006-01-01

A method for improving the identification of peptides in a shotgun proteome analysis using accurate mass measurement has been developed. The improvement is based upon the derivatization of cysteine residues with a novel reagent, 2,4-dibromo-(2′-iodo)acetanilide. The derivitization changes the mass defect of cysteine-containing proteolytic peptides in a manner that increases their identification specificity. Peptide masses were measured using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron mass spectrometry. Reactions with protein standards show that the derivatization of cysteine is rapid and quantitative, and the data suggest that the derivatized peptides are more easily ionized or detected than unlabeled cysteine-containing peptides. The reagent was tested on a 15N-metabolically labeled proteome from M. maripaludis. Proteins were identified by their accurate mass values and from their nitrogen stoichiometry. A total of 47% of the labeled peptides are identified versus 27% for the unlabeled peptides. This procedure permits the identification of proteins from the M. maripaludis proteome that are not usually observed by the standard protocol and shows that better protein coverage is obtained with this methodology. PMID:16689545

Understanding global changes of the liver proteome during murine schistosomiasis using a label-free shotgun approach.

PubMed

Campos, Jonatan Marques; Neves, Leandro Xavier; de Paiva, Nívia Carolina Nogueira; de Oliveira E Castro, Renata Alves; Casé, Ana Helena; Carneiro, Cláudia Martins; Andrade, Milton Hércules Guerra; Castro-Borges, William

2017-01-16

Schistosomiasis is an endemic disease affecting over 207 million people worldwide caused by helminth parasites of the genus Schistosoma. In Brazil the disease is responsible for the loss of up to 800 lives annually, resulting from the desabilitating effects of this chronic condition. In this study, we infected Balb/c mice with Schistosoma mansoni and analysed global changes in the proteomic profile of soluble liver proteins. Our shotgun analyses revealed predominance of up-regulation of proteins at 5weeks of infection, coinciding with the onset of egg laying, and a remarkable down-regulation of liver constituents at 7weeks, when severe tissue damage is installed. Representatives of glycolytic enzymes and stress response (in particular at the endoplasmic reticulum) were among the most differentially expressed molecules found in the infected liver. Collectively, our data contribute over 70 molecules not previously reported to be found at altered levels in murine schistosomiasis to further exploration of their potential as biomarkers of the disease. Moreover, understanding their intricate interaction using bioinformatics approach can potentially bring clarity to unknown mechanisms linked to the establishment of this condition in the vertebrate host. To our knowledge, this study refers to the first shotgun proteomic analysis to provide an inventory of the global changes in the liver soluble proteome caused by Schistosoma mansoni in the Balb/c model. It also innovates by yielding data on quantification of the identified molecules as a manner to clarify and give insights into the underlying mechanisms for establishment of Schistosomiasis, a neglected tropical disease with historical prevalence in Brazil. Copyright © 2016 Elsevier B.V. All rights reserved.

Soup to Tree: The Phylogeny of Beetles Inferred by Mitochondrial Metagenomics of a Bornean Rainforest Sample.

PubMed

Crampton-Platt, Alex; Timmermans, Martijn J T N; Gimmel, Matthew L; Kutty, Sujatha Narayanan; Cockerill, Timothy D; Vun Khen, Chey; Vogler, Alfried P

2015-09-01

In spite of the growth of molecular ecology, systematics and next-generation sequencing, the discovery and analysis of diversity is not currently integrated with building the tree-of-life. Tropical arthropod ecologists are well placed to accelerate this process if all specimens obtained through mass-trapping, many of which will be new species, could be incorporated routinely into phylogeny reconstruction. Here we test a shotgun sequencing approach, whereby mitochondrial genomes are assembled from complex ecological mixtures through mitochondrial metagenomics, and demonstrate how the approach overcomes many of the taxonomic impediments to the study of biodiversity. DNA from approximately 500 beetle specimens, originating from a single rainforest canopy fogging sample from Borneo, was pooled and shotgun sequenced, followed by de novo assembly of complete and partial mitogenomes for 175 species. The phylogenetic tree obtained from this local sample was highly similar to that from existing mitogenomes selected for global coverage of major lineages of Coleoptera. When all sequences were combined only minor topological changes were induced against this reference set, indicating an increasingly stable estimate of coleopteran phylogeny, while the ecological sample expanded the tip-level representation of several lineages. Robust trees generated from ecological samples now enable an evolutionary framework for ecology. Meanwhile, the inclusion of uncharacterized samples in the tree-of-life rapidly expands taxon and biogeographic representation of lineages without morphological identification. Mitogenomes from shotgun sequencing of unsorted environmental samples and their associated metadata, placed robustly into the phylogenetic tree, constitute novel DNA "superbarcodes" for testing hypotheses regarding global patterns of diversity. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Use of Metagenomic Shotgun Sequencing Technology To Detect Foodborne Pathogens within the Microbiome of the Beef Production Chain.

PubMed

Yang, Xiang; Noyes, Noelle R; Doster, Enrique; Martin, Jennifer N; Linke, Lyndsey M; Magnuson, Roberta J; Yang, Hua; Geornaras, Ifigenia; Woerner, Dale R; Jones, Kenneth L; Ruiz, Jaime; Boucher, Christina; Morley, Paul S; Belk, Keith E

2016-04-01

Foodborne illnesses associated with pathogenic bacteria are a global public health and economic challenge. The diversity of microorganisms (pathogenic and nonpathogenic) that exists within the food and meat industries complicates efforts to understand pathogen ecology. Further, little is known about the interaction of pathogens within the microbiome throughout the meat production chain. Here, a metagenomic approach and shotgun sequencing technology were used as tools to detect pathogenic bacteria in environmental samples collected from the same groups of cattle at different longitudinal processing steps of the beef production chain: cattle entry to feedlot, exit from feedlot, cattle transport trucks, abattoir holding pens, and the end of the fabrication system. The log read counts classified as pathogens per million reads for Salmonella enterica,Listeria monocytogenes,Escherichia coli,Staphylococcus aureus, Clostridium spp. (C. botulinum and C. perfringens), and Campylobacter spp. (C. jejuni,C. coli, and C. fetus) decreased over subsequential processing steps. Furthermore, the normalized read counts for S. enterica,E. coli, and C. botulinumwere greater in the final product than at the feedlots, indicating that the proportion of these bacteria increased (the effect on absolute numbers was unknown) within the remaining microbiome. From an ecological perspective, data indicated that shotgun metagenomics can be used to evaluate not only the microbiome but also shifts in pathogen populations during beef production. Nonetheless, there were several challenges in this analysis approach, one of the main ones being the identification of the specific pathogen from which the sequence reads originated, which makes this approach impractical for use in pathogen identification for regulatory and confirmation purposes. Copyright © 2016 Yang et al.

Shotgun proteomic analysis of Emiliania huxleyi, a marine phytoplankton species of major biogeochemical importance.

PubMed

Jones, Bethan M; Edwards, Richard J; Skipp, Paul J; O'Connor, C David; Iglesias-Rodriguez, M Debora

2011-06-01

Emiliania huxleyi is a unicellular marine phytoplankton species known to play a significant role in global biogeochemistry. Through the dual roles of photosynthesis and production of calcium carbonate (calcification), carbon is transferred from the atmosphere to ocean sediments. Almost nothing is known about the molecular mechanisms that control calcification, a process that is tightly regulated within the cell. To initiate proteomic studies on this important and phylogenetically remote organism, we have devised efficient protein extraction protocols and developed a bioinformatics pipeline that allows the statistically robust assignment of proteins from MS/MS data using preexisting EST sequences. The bioinformatics tool, termed BUDAPEST (Bioinformatics Utility for Data Analysis of Proteomics using ESTs), is fully automated and was used to search against data generated from three strains. BUDAPEST increased the number of identifications over standard protein database searches from 37 to 99 proteins when data were amalgamated. Proteins involved in diverse cellular processes were uncovered. For example, experimental evidence was obtained for a novel type I polyketide synthase and for various photosystem components. The proteomic and bioinformatic approaches developed in this study are of wider applicability, particularly to the oceanographic community where genomic sequence data for species of interest are currently scarce.

DAnTE: a statistical tool for quantitative analysis of –omics data

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

Polpitiya, Ashoka D.; Qian, Weijun; Jaitly, Navdeep

2008-05-03

DAnTE (Data Analysis Tool Extension) is a statistical tool designed to address challenges unique to quantitative bottom-up, shotgun proteomics data. This tool has also been demonstrated for microarray data and can easily be extended to other high-throughput data types. DAnTE features selected normalization methods, missing value imputation algorithms, peptide to protein rollup methods, an extensive array of plotting functions, and a comprehensive ANOVA scheme that can handle unbalanced data and random effects. The Graphical User Interface (GUI) is designed to be very intuitive and user friendly.

Gene Expression Analysis of CL-20-induced Reversible Neurotoxicity Reveals GABAA Receptors as Potential Target in the Earthworm Eisenia fetida

PubMed Central

Gong, Ping; Guan, Xin; Pirooznia, Mehdi; Liang, Chun; Perkins, Edward J.

2012-01-01

The earthworm Eisenia fetida is one of the most used species in standardized soil ecotoxicity tests. Endpoints such as survival, growth and reproduction are eco-toxicologically relevant but provide little mechanistic insight into toxicity pathways, especially at the molecular level. Here we applied a toxicogenomic approach to investigate the mode of action underlying the reversible neurotoxicity of hexanitrohexaazaisowurtzitane (CL-20), a cyclic nitroamine explosives compound. We developed an E. fetida-specific shotgun microarray targeting 15119 unique E. fetida transcripts. Using this array we profiled gene expression in E. fetida in response to exposure to CL-20. Eighteen earthworms were exposed for 6 days to 0.2 μg/cm2 of CL-20 on filter paper, half of which were allowed to recover in a clean environment for 7 days. Nine vehicle control earthworms were sacrificed at day 6 and 13, separately. Electrophysiological measurements indicated that the conduction velocity of earthworm medial giant nerve fiber decreased significantly after 6-day exposure to CL-20, but was restored after 7 days of recovery. Total RNA was isolated from the four treatment groups including 6-day control, 6-day exposed, 13-day control and 13-day exposed (i.e. 6-day exposure followed by 7-day recovery), and was hybridized to the 15K shot-gun oligo array. Statistical and bioinformatic analyses suggest that CL-20 initiated neurotoxicity by non-competitively blocking the ligand-gated GABAA receptor ion channel, leading to altered expression of genes involved in GABAergic, cholinergic, and Agrin-MuSK pathways. In the recovery phase, expression of affected genes returned to normality, possibly as a result of autophagy and CL-20 dissociation/metabolism. This study provides significant insights into potential mechanisms of CL-20-induced neurotoxicity and the recovery of earthworms from transient neurotoxicity stress. PMID:22191394

Systematic Evaluation of the Use of Human Plasma and Serum for Mass-Spectrometry-Based Shotgun Proteomics.

PubMed

Lan, Jiayi; Núñez Galindo, Antonio; Doecke, James; Fowler, Christopher; Martins, Ralph N; Rainey-Smith, Stephanie R; Cominetti, Ornella; Dayon, Loïc

2018-04-06

Over the last two decades, EDTA-plasma has been used as the preferred sample matrix for human blood proteomic profiling. Serum has also been employed widely. Only a few studies have assessed the difference and relevance of the proteome profiles obtained from plasma samples, such as EDTA-plasma or lithium-heparin-plasma, and serum. A more complete evaluation of the use of EDTA-plasma, heparin-plasma, and serum would greatly expand the comprehensiveness of shotgun proteomics of blood samples. In this study, we evaluated the use of heparin-plasma with respect to EDTA-plasma and serum to profile blood proteomes using a scalable automated proteomic pipeline (ASAP 2 ). The use of plasma and serum for mass-spectrometry-based shotgun proteomics was first tested with commercial pooled samples. The proteome coverage consistency and the quantitative performance were compared. Furthermore, protein measurements in EDTA-plasma and heparin-plasma samples were comparatively studied using matched sample pairs from 20 individuals from the Australian Imaging, Biomarkers and Lifestyle (AIBL) Study. We identified 442 proteins in common between EDTA-plasma and heparin-plasma samples. Overall agreement of the relative protein quantification between the sample pairs demonstrated that shotgun proteomics using workflows such as the ASAP 2 is suitable in analyzing heparin-plasma and that such sample type may be considered in large-scale clinical research studies. Moreover, the partial proteome coverage overlaps (e.g., ∼70%) showed that measures from heparin-plasma could be complementary to those obtained from EDTA-plasma.

Mid-Term Outcomes After Open Reduction Internal Fixation of Proximal Interphalangeal Joint Dorsal Fracture-Dislocations Through a Volar, Shotgun Approach and a Review of the Literature

PubMed Central

Giugale, Juan Marcelo; Wang, Juntian; Kaufmann, Robert A.; Fowler, John R.

2017-01-01

Background: Proximal interphalangeal (PIP) fracture dislocations remain a complex injury pattern to treat. There are several treatment methods available aimed to restore stability, preserve range of motion, and reconstitute the articular surface. This study looked at the mid-term clinical and radiographic results of open reduction internal fixation through a shotgun approach of comminuted PIP fracture dislocations. Methods: A retrospective review was conducted of all PIP fracture dislocations treated through a volar, shotgun approach at a single institution over a 15-year period. Patients identified were contacted and asked to return to the office for clinical and radiographic evaluation. Patient reported outcomes were assessed with the Michigan hand questionnaire (MHQ) and visual analog scale (VAS) for pain. Results: 5 patients returned to the office for further evaluation with average follow-up of 69 months (range, 33-133 months). 3 patients were found to have post traumatic arthritis on radiographs. 1 case had recurrent instability and one case had a deep infection, both necessitating further surgical intervention. Average PIP arc of motion was found to be 79°. Average VAS score of 0 and MHQ result of 95 (out of a possible score of 100) indicating no residual pain and excellent functionality of the affected hand. Conclusion: Open reduction internal fixation of comminuted PIP fracture dislocations utilizing the volar, shotgun approach provides excellent mid-term functional results despite the high incidence of post traumatic arthritis. PMID:29151999

Capturing chloroplast variation for molecular ecology studies: a simple next generation sequencing approach applied to a rainforest tree

PubMed Central

2013-01-01

Background With high quantity and quality data production and low cost, next generation sequencing has the potential to provide new opportunities for plant phylogeographic studies on single and multiple species. Here we present an approach for in silicio chloroplast DNA assembly and single nucleotide polymorphism detection from short-read shotgun sequencing. The approach is simple and effective and can be implemented using standard bioinformatic tools. Results The chloroplast genome of Toona ciliata (Meliaceae), 159,514 base pairs long, was assembled from shotgun sequencing on the Illumina platform using de novo assembly of contigs. To evaluate its practicality, value and quality, we compared the short read assembly with an assembly completed using 454 data obtained after chloroplast DNA isolation. Sanger sequence verifications indicated that the Illumina dataset outperformed the longer read 454 data. Pooling of several individuals during preparation of the shotgun library enabled detection of informative chloroplast SNP markers. Following validation, we used the identified SNPs for a preliminary phylogeographic study of T. ciliata in Australia and to confirm low diversity across the distribution. Conclusions Our approach provides a simple method for construction of whole chloroplast genomes from shotgun sequencing of whole genomic DNA using short-read data and no available closely related reference genome (e.g. from the same species or genus). The high coverage of Illumina sequence data also renders this method appropriate for multiplexing and SNP discovery and therefore a useful approach for landscape level studies of evolutionary ecology. PMID:23497206

A new type of shotgun ammunition produces unique wound characteristics.

PubMed

Nelson, Craig L; Winston, David C

2007-01-01

The Tucson Police Department, Tucson, AZ, has begun using the Polyshok Impact Reactive Projectile (IRP), a new type of shotgun ammunition that includes a lead bead core that travels within single, plastic-encased projectile. On impact, the core is released to distribute over a small area, thereby disintegrating on impact to reduce the likelihood of exit or collateral damage on missed shots. After a brief review of shotgun slug ballistics and wound characteristics and a discussion of the mechanism of the Polyshok IRP, we report the first death in the United States from this ammunition. Findings included a single entrance wound with plastic ammunition components and small lead particles recovered from the body, the combination of which normally would suggest a close-range shooting with birdshot. However, the characteristics of this ammunition create different patterns than are found with slugs or shot, so that a medical examiner unfamiliar with the Polyshok IRP could draw inaccurate conclusions about ammunition and range of fire. Because the single projectile fired from this ammunition is composed of both plastic and lead, plastic components are likely to be found within the wound at any range of fire, unlike traditional shot or slug ammunition. Also, the small size of lead particles found spread through the wound cavity would ordinarily suggest a small-size shot, whereas the external appearance of the wound (a single entrance with no dispersion of shot) and the pattern of tissue destruction are more consistent with the patterns of injury associated with shotgun slugs.

Extending the Limits of Quantitative Proteome Profiling with Data-Independent Acquisition and Application to Acetaminophen-Treated Three-Dimensional Liver Microtissues*

PubMed Central

Bruderer, Roland; Bernhardt, Oliver M.; Gandhi, Tejas; Miladinović, Saša M.; Cheng, Lin-Yang; Messner, Simon; Ehrenberger, Tobias; Zanotelli, Vito; Butscheid, Yulia; Escher, Claudia; Vitek, Olga; Rinner, Oliver; Reiter, Lukas

2015-01-01

The data-independent acquisition (DIA) approach has recently been introduced as a novel mass spectrometric method that promises to combine the high content aspect of shotgun proteomics with the reproducibility and precision of selected reaction monitoring. Here, we evaluate, whether SWATH-MS type DIA effectively translates into a better protein profiling as compared with the established shotgun proteomics. We implemented a novel DIA method on the widely used Orbitrap platform and used retention-time-normalized (iRT) spectral libraries for targeted data extraction using Spectronaut. We call this combination hyper reaction monitoring (HRM). Using a controlled sample set, we show that HRM outperformed shotgun proteomics both in the number of consistently identified peptides across multiple measurements and quantification of differentially abundant proteins. The reproducibility of HRM in peptide detection was above 98%, resulting in quasi complete data sets compared with 49% of shotgun proteomics. Utilizing HRM, we profiled acetaminophen (APAP)1-treated three-dimensional human liver microtissues. An early onset of relevant proteome changes was revealed at subtoxic doses of APAP. Further, we detected and quantified for the first time human NAPQI-protein adducts that might be relevant for the toxicity of APAP. The adducts were identified on four mitochondrial oxidative stress related proteins (GATM, PARK7, PRDX6, and VDAC2) and two other proteins (ANXA2 and FTCD). Our findings imply that DIA should be the preferred method for quantitative protein profiling. PMID:25724911

GO Explorer: A gene-ontology tool to aid in the interpretation of shotgun proteomics data.

PubMed

Carvalho, Paulo C; Fischer, Juliana Sg; Chen, Emily I; Domont, Gilberto B; Carvalho, Maria Gc; Degrave, Wim M; Yates, John R; Barbosa, Valmir C

2009-02-24

Spectral counting is a shotgun proteomics approach comprising the identification and relative quantitation of thousands of proteins in complex mixtures. However, this strategy generates bewildering amounts of data whose biological interpretation is a challenge. Here we present a new algorithm, termed GO Explorer (GOEx), that leverages the gene ontology (GO) to aid in the interpretation of proteomic data. GOEx stands out because it combines data from protein fold changes with GO over-representation statistics to help draw conclusions. Moreover, it is tightly integrated within the PatternLab for Proteomics project and, thus, lies within a complete computational environment that provides parsers and pattern recognition tools designed for spectral counting. GOEx offers three independent methods to query data: an interactive directed acyclic graph, a specialist mode where key words can be searched, and an automatic search. Its usefulness is demonstrated by applying it to help interpret the effects of perillyl alcohol, a natural chemotherapeutic agent, on glioblastoma multiform cell lines (A172). We used a new multi-surfactant shotgun proteomic strategy and identified more than 2600 proteins; GOEx pinpointed key sets of differentially expressed proteins related to cell cycle, alcohol catabolism, the Ras pathway, apoptosis, and stress response, to name a few. GOEx facilitates organism-specific studies by leveraging GO and providing a rich graphical user interface. It is a simple to use tool, specialized for biologists who wish to analyze spectral counting data from shotgun proteomics. GOEx is available at http://pcarvalho.com/patternlab.

Unveiling the gut microbiota composition and functionality associated with constipation through metagenomic analyses.

PubMed

Mancabelli, Leonardo; Milani, Christian; Lugli, Gabriele Andrea; Turroni, Francesca; Mangifesta, Marta; Viappiani, Alice; Ticinesi, Andrea; Nouvenne, Antonio; Meschi, Tiziana; van Sinderen, Douwe; Ventura, Marco

2017-08-29

Functional constipation (FC) is a gastrointestinal disorder with a high prevalence among the general population. The precise causes of FC are still unknown and are most likely multifactorial. Growing evidence indicates that alterations of gut microbiota composition contribute to constipation symptoms. Nevertheless, many discrepancies exist in literature and no clear link between FC and gut microbiota composition has as yet been identified. In this study, we performed 16 S rRNA-based microbial profiling analysis of 147 stool samples from 68 FC individuals and compared their microbial profiles with those of 79 healthy subjects (HS). Notably, the gut microbiota of FC individuals was shown to be depleted of members belonging to Bacteroides, Roseburia and Coprococcus 3. Furthermore, the metabolic capabilities of the gut microbiomes of five FC and five HS individuals were evaluated through shotgun metagenomics using a MiSeq platform, indicating that HS are enriched in pathways involved in carbohydrate, fatty acid and lipid metabolism as compared to FC. In contrast, the microbiomes corresponding to FC were shown to exhibit high abundance of genes involved in hydrogen production, methanogenesis and glycerol degradation. The identified differences in bacterial composition and metabolic capabilities may play an important role in development of FC symptoms.

[The possibility of using PlasmaDeepDive™ MRM panel in clinical diagnostics].

PubMed

Miroshnichenko, Iu V; Petushkova, N A; Moskaleva, N E; Teryaeva, N B; Zgoda, V G; Ilgisonis, E V; Belyaev, A Yu

2015-01-01

Concentrations of 46 proteins have been determined in human blood plasma using PlasmaDeepDive™ MRM Panel ("Biognosys AG", Switzerland). 18 of them were included into the group of proteins with higher concentrations, also identified by the shotgun proteomic analysis. Based on literature data it is concluded that the PlasmaDeepDive™ MRM Panel is applicable for studies of human plasma samples for potential biomarkers of various nervous system disorders.

Standard Scenarios for the Less-Lethal Weapons Evaluation Model

DTIC Science & Technology

1975-08-01

smoke (for obscuration) with chemical irritant. "MODIPAC" - 12 - gauge plastic shotgun shell loaded with polyethyleie shot. Net - A net for entrapment...Oklahoma, U. S. Department of Justice, April 30, 1974 (Grant Numbers 73-TA-08-0004 and 73-DF-06-0053). 12 Ana~lysis of Scenarios a. The developmecnt of...for transmitting electrical energy (e.g., TASER ). Electrical Prod - A device to transmit electrical shock to the target. Heavy Slug - Experimental

Salt stress induces changes in the proteomic profile of micropropagated sugarcane shoots

PubMed Central

Reis, Ricardo S.; Heringer, Angelo S.; Rangel, Patricia L.; Santa-Catarina, Claudete; Grativol, Clícia; Veiga, Carlos F. M.; Souza-Filho, Gonçalo A.

2017-01-01

Salt stress is one of the most common stresses in agricultural regions worldwide. In particular, sugarcane is affected by salt stress conditions, and no sugarcane cultivar presently show high productivity accompanied by a tolerance to salt stress. Proteomic analysis allows elucidation of the important pathways involved in responses to various abiotic stresses at the biochemical and molecular levels. Thus, this study aimed to analyse the proteomic effects of salt stress in micropropagated shoots of two sugarcane cultivars (CB38-22 and RB855536) using a label-free proteomic approach. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD006075. The RB855536 cultivar is more tolerant to salt stress than CB38-22. A quantitative label-free shotgun proteomic analysis identified 1172 non-redundant proteins, and 1160 of these were observed in both cultivars in the presence or absence of NaCl. Compared with CB38-22, the RB855536 cultivar showed a greater abundance of proteins involved in non-enzymatic antioxidant mechanisms, ion transport, and photosynthesis. Some proteins, such as calcium-dependent protein kinase, photosystem I, phospholipase D, and glyceraldehyde-3-phosphate dehydrogenase, were more abundant in the RB855536 cultivar under salt stress. Our results provide new insights into the response of sugarcane to salt stress, and the changes in the abundance of these proteins might be important for the acquisition of ionic and osmotic homeostasis during exposure to salt stress. PMID:28419154

A combined de novo protein sequencing and cDNA library approach to the venomic analysis of Chinese spider Araneus ventricosus.

PubMed

Duan, Zhigui; Cao, Rui; Jiang, Liping; Liang, Songping

2013-01-14

In past years, spider venoms have attracted increasing attention due to their extraordinary chemical and pharmacological diversity. The recently popularized proteomic method highly improved our ability to analyze the proteins in the venom. However, the lack of information about isolated venom proteins sequences dramatically limits the ability to confidently identify venom proteins. In the present paper, the venom from Araneus ventricosus was analyzed using two complementary approaches: 2-DE/Shotgun-LC-MS/MS coupled to MASCOT search and 2-DE/Shotgun-LC-MS/MS coupled to manual de novo sequencing followed by local venom protein database (LVPD) search. The LVPD was constructed with toxin-like protein sequences obtained from the analysis of cDNA library from A. ventricosus venom glands. Our results indicate that a total of 130 toxin-like protein sequences were unambiguously identified by manual de novo sequencing coupled to LVPD search, accounting for 86.67% of all toxin-like proteins in LVPD. Thus manual de novo sequencing coupled to LVPD search was proved an extremely effective approach for the analysis of venom proteins. In addition, the approach displays impeccable advantage in validating mutant positions of isoforms from the same toxin-like family. Intriguingly, methyl esterifcation of glutamic acid was discovered for the first time in animal venom proteins by manual de novo sequencing. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

Taxonomic and Functional Microbial Signatures of the Endemic Marine Sponge Arenosclera brasiliensis

PubMed Central

Trindade-Silva, Amaro E.; Rua, Cintia; Silva, Genivaldo G. Z.; Dutilh, Bas E.; Moreira, Ana Paula B.; Edwards, Robert A.; Hajdu, Eduardo; Lobo-Hajdu, Gisele; Vasconcelos, Ana Tereza; Berlinck, Roberto G. S.; Thompson, Fabiano L.

2012-01-01

The endemic marine sponge Arenosclera brasiliensis (Porifera, Demospongiae, Haplosclerida) is a known source of secondary metabolites such as arenosclerins A-C. In the present study, we established the composition of the A. brasiliensis microbiome and the metabolic pathways associated with this community. We used 454 shotgun pyrosequencing to generate approximately 640,000 high-quality sponge-derived sequences (∼150 Mb). Clustering analysis including sponge, seawater and twenty-three other metagenomes derived from marine animal microbiomes shows that A. brasiliensis contains a specific microbiome. Fourteen bacterial phyla (including Proteobacteria, Cyanobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Cloroflexi) were consistently found in the A. brasiliensis metagenomes. The A. brasiliensis microbiome is enriched for Betaproteobacteria (e.g., Burkholderia) and Gammaproteobacteria (e.g., Pseudomonas and Alteromonas) compared with the surrounding planktonic microbial communities. Functional analysis based on Rapid Annotation using Subsystem Technology (RAST) indicated that the A. brasiliensis microbiome is enriched for sequences associated with membrane transport and one-carbon metabolism. In addition, there was an overrepresentation of sequences associated with aerobic and anaerobic metabolism as well as the synthesis and degradation of secondary metabolites. This study represents the first analysis of sponge-associated microbial communities via shotgun pyrosequencing, a strategy commonly applied in similar analyses in other marine invertebrate hosts, such as corals and algae. We demonstrate that A. brasiliensis has a unique microbiome that is distinct from that of the surrounding planktonic microbes and from other marine organisms, indicating a species-specific microbiome. PMID:22768320

Tandem Mass Spectrum Sequencing: An Alternative to Database Search Engines in Shotgun Proteomics.

PubMed

Muth, Thilo; Rapp, Erdmann; Berven, Frode S; Barsnes, Harald; Vaudel, Marc

2016-01-01

Protein identification via database searches has become the gold standard in mass spectrometry based shotgun proteomics. However, as the quality of tandem mass spectra improves, direct mass spectrum sequencing gains interest as a database-independent alternative. In this chapter, the general principle of this so-called de novo sequencing is introduced along with pitfalls and challenges of the technique. The main tools available are presented with a focus on user friendly open source software which can be directly applied in everyday proteomic workflows.

Archeological Testing of the North Bend Site (16SMY132) and Survey of the Todd Area Levee, St. Mary Parish, Louisiana

DTIC Science & Technology

1993-07-01

borings and from the drilling of vibracore or hand auger holes for a specific project. The terrane is the unit used to map the subsurface distribution...three pieces of amethyst glass and a piece of dark green bottle glass. Two pieces of coal, one indeterminate nail fragment and a 12- gauge shotgun shell...and three pieces of indeterminate iron. Also recovered were a 12- gauge shotgun shell base, and one brick fragment. Based on the stratigraphy all were

Feedback Microtubule Control and Microtubule-Actin Cross-talk in Arabidopsis Revealed by Integrative Proteomic and Cell Biology Analysis of KATANIN 1 Mutants.

PubMed

Takáč, Tomáš; Šamajová, Olga; Pechan, Tibor; Luptovčiak, Ivan; Šamaj, Jozef

2017-09-01

Microtubule organization and dynamics are critical for key developmental processes such as cell division, elongation, and morphogenesis. Microtubule severing is an essential regulator of microtubules and is exclusively executed by KATANIN 1 in Arabidopsis In this study, we comparatively studied the proteome-wide effects in two KATANIN 1 mutants. Thus, shotgun proteomic analysis of roots and aerial parts of single nucleotide mutant fra2 and T-DNA insertion mutant ktn1-2 was carried out. We have detected 42 proteins differentially abundant in both fra2 and ktn1-2 KATANIN 1 dysfunction altered the abundance of proteins involved in development, metabolism, and stress responses. The differential regulation of tubulins and microtubule-destabilizing protein MDP25 implied a feedback microtubule control in KATANIN 1 mutants. Furthermore, deregulation of profilin 1, actin-depolymerizing factor 3, and actin 7 was observed. These findings were confirmed by immunoblotting analysis of actin and by microscopic observation of actin filaments using fluorescently labeled phalloidin. Results obtained by quantitative RT-PCR analysis revealed that changed protein abundances were not a consequence of altered expression levels of corresponding genes in the mutants. In conclusion, we show that abundances of several cytoskeletal proteins as well as organization of microtubules and the actin cytoskeleton are amended in accordance with defective microtubule severing. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The MaxQuant computational platform for mass spectrometry-based shotgun proteomics.

PubMed

Tyanova, Stefka; Temu, Tikira; Cox, Juergen

2016-12-01

MaxQuant is one of the most frequently used platforms for mass-spectrometry (MS)-based proteomics data analysis. Since its first release in 2008, it has grown substantially in functionality and can be used in conjunction with more MS platforms. Here we present an updated protocol covering the most important basic computational workflows, including those designed for quantitative label-free proteomics, MS1-level labeling and isobaric labeling techniques. This protocol presents a complete description of the parameters used in MaxQuant, as well as of the configuration options of its integrated search engine, Andromeda. This protocol update describes an adaptation of an existing protocol that substantially modifies the technique. Important concepts of shotgun proteomics and their implementation in MaxQuant are briefly reviewed, including different quantification strategies and the control of false-discovery rates (FDRs), as well as the analysis of post-translational modifications (PTMs). The MaxQuant output tables, which contain information about quantification of proteins and PTMs, are explained in detail. Furthermore, we provide a short version of the workflow that is applicable to data sets with simple and standard experimental designs. The MaxQuant algorithms are efficiently parallelized on multiple processors and scale well from desktop computers to servers with many cores. The software is written in C# and is freely available at http://www.maxquant.org.

Cost-effective sequencing of full-length cDNA clones powered by a de novo-reference hybrid assembly.

PubMed

Kuroshu, Reginaldo M; Watanabe, Junichi; Sugano, Sumio; Morishita, Shinichi; Suzuki, Yutaka; Kasahara, Masahiro

2010-05-07

Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded. We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence approximately 800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species. The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only approximately US$3 per clone, demonstrating a significant advantage over previous approaches.

A proteomic insight into vitellogenesis during tick ovary maturation.

PubMed

Xavier, Marina Amaral; Tirloni, Lucas; Pinto, Antônio F M; Diedrich, Jolene K; Yates, John R; Mulenga, Albert; Logullo, Carlos; da Silva Vaz, Itabajara; Seixas, Adriana; Termignoni, Carlos

2018-03-16

Ticks are arthropod ectoparasites of importance for public and veterinary health. The understanding of tick oogenesis and embryogenesis could contribute to the development of novel control methods. However, to date, studies on the temporal dynamics of proteins during ovary development were not reported. In the present study we followed protein profile during ovary maturation. Proteomic analysis of ovary extracts was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using shotgun strategy, in addition to dimethyl labelling-based protein quantification. A total of 3,756 proteins were identified, which were functionally annotated into 30 categories. Circa 80% of the annotated proteins belong to categories related to basal metabolism, such as protein synthesis and modification machineries, nuclear regulation, cytoskeleton, proteasome machinery, transcriptional machinery, energetic metabolism, extracellular matrix/cell adhesion, immunity, oxidation/detoxification metabolism, signal transduction, and storage. The abundance of selected proteins involved in yolk uptake and degradation, as well as vitellin accumulation during ovary maturation, was assessed using dimethyl-labelling quantification. In conclusion, proteins identified in this study provide a framework for future studies to elucidate tick development and validate candidate targets for novel control methods.

Phylogenetic screening of a bacterial, metagenomic library using homing endonuclease restriction and marker insertion

PubMed Central

Yung, Pui Yi; Burke, Catherine; Lewis, Matt; Egan, Suhelen; Kjelleberg, Staffan; Thomas, Torsten

2009-01-01

Metagenomics provides access to the uncultured majority of the microbial world. The approaches employed in this field have, however, had limited success in linking functional genes to the taxonomic or phylogenetic origin of the organism they belong to. Here we present an efficient strategy to recover environmental DNA fragments that contain phylogenetic marker genes from metagenomic libraries. Our method involves the cleavage of 23S ribsosmal RNA (rRNA) genes within pooled library clones by the homing endonuclease I-CeuI followed by the insertion and selection of an antibiotic resistance cassette. This approach was applied to screen a library of 6500 fosmid clones derived from the microbial community associated with the sponge Cymbastela concentrica. Several fosmid clones were recovered after the screen and detailed phylogenetic and taxonomic assignment based on the rRNA gene showed that they belong to previously unknown organisms. In addition, compositional features of these fosmid clones were used to classify and taxonomically assign a dataset of environmental shotgun sequences. Our approach represents a valuable tool for the analysis of rapidly increasing, environmental DNA sequencing information. PMID:19767618

A review of lipidomic technologies applicable to sphingolipidomics and their relevant applications

PubMed Central

Han, Xianlin; Jiang, Xuntian

2009-01-01

Sphingolipidomics, a branch of lipidomics, focuses on the large-scale study of the cellular sphingolipidomes. In the current review, two main approaches for the analysis of cellular sphingolipidomes (i.e. LC-MS- or LC-MS/MS-based approach and shotgun lipidomics-based approach) are briefly discussed. Their advantages, some considerations of these methods, and recent applications of these approaches are summarized. It is the authors’ sincere hope that this review article will add to the readers understanding of the advantages and limitations of each developed method for the analysis of a cellular sphingolipidome. PMID:19690629

N-linked glycoprotein analysis using dual-extraction ultrahigh-performance liquid chromatography and electrospray tandem mass spectrometry.

PubMed

Siu, S O; Lam, Maggie P Y; Lau, Edward; Yeung, William S B; Cox, David M; Chu, Ivan K

2010-01-01

Although reverse-phase liquid chromatography (RP-LC) is a common technique for peptide separation in shotgun proteomics and glycoproteomics, it often provides unsatisfactory results for the analysis of glycopeptides and glycans. This bias against glycopeptides makes it difficult to study glycoproteins. By coupling mass spectrometry (MS) with a combination of RP-LC and normal-phase (NP)-LC as an integrated front-end separation system, we demonstrate that effective identification and characterization of both peptides and glycopeptides mixtures, and their constituent glycan structures, can be achieved from a single sample injection event.

Identification of fungi in shotgun metagenomics datasets

PubMed Central

Donovan, Paul D.; Gonzalez, Gabriel; Higgins, Desmond G.

2018-01-01

Metagenomics uses nucleic acid sequencing to characterize species diversity in different niches such as environmental biomes or the human microbiome. Most studies have used 16S rRNA amplicon sequencing to identify bacteria. However, the decreasing cost of sequencing has resulted in a gradual shift away from amplicon analyses and towards shotgun metagenomic sequencing. Shotgun metagenomic data can be used to identify a wide range of species, but have rarely been applied to fungal identification. Here, we develop a sequence classification pipeline, FindFungi, and use it to identify fungal sequences in public metagenome datasets. We focus primarily on animal metagenomes, especially those from pig and mouse microbiomes. We identified fungi in 39 of 70 datasets comprising 71 fungal species. At least 11 pathogenic species with zoonotic potential were identified, including Candida tropicalis. We identified Pseudogymnoascus species from 13 Antarctic soil samples initially analyzed for the presence of bacteria capable of degrading diesel oil. We also show that Candida tropicalis and Candida loboi are likely the same species. In addition, we identify several examples where contaminating DNA was erroneously included in fungal genome assemblies. PMID:29444186

SUPER-FOCUS: a tool for agile functional analysis of shotgun metagenomic data

PubMed Central

Green, Kevin T.; Dutilh, Bas E.; Edwards, Robert A.

2016-01-01

Summary: Analyzing the functional profile of a microbial community from unannotated shotgun sequencing reads is one of the important goals in metagenomics. Functional profiling has valuable applications in biological research because it identifies the abundances of the functional genes of the organisms present in the original sample, answering the question what they can do. Currently, available tools do not scale well with increasing data volumes, which is important because both the number and lengths of the reads produced by sequencing platforms keep increasing. Here, we introduce SUPER-FOCUS, SUbsystems Profile by databasE Reduction using FOCUS, an agile homology-based approach using a reduced reference database to report the subsystems present in metagenomic datasets and profile their abundances. SUPER-FOCUS was tested with over 70 real metagenomes, the results showing that it accurately predicts the subsystems present in the profiled microbial communities, and is up to 1000 times faster than other tools. Availability and implementation: SUPER-FOCUS was implemented in Python, and its source code and the tool website are freely available at https://edwards.sdsu.edu/SUPERFOCUS. Contact: redwards@mail.sdsu.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26454280

Large-scale human skin lipidomics by quantitative, high-throughput shotgun mass spectrometry.

PubMed

Sadowski, Tomasz; Klose, Christian; Gerl, Mathias J; Wójcik-Maciejewicz, Anna; Herzog, Ronny; Simons, Kai; Reich, Adam; Surma, Michal A

2017-03-07

The lipid composition of human skin is essential for its function; however the simultaneous quantification of a wide range of stratum corneum (SC) and sebaceous lipids is not trivial. We developed and validated a quantitative high-throughput shotgun mass spectrometry-based platform for lipid analysis of tape-stripped SC skin samples. It features coverage of 16 lipid classes; total quantification to the level of individual lipid molecules; high reproducibility and high-throughput capabilities. With this method we conducted a large lipidomic survey of 268 human SC samples, where we investigated the relationship between sampling depth and lipid composition, lipidome variability in samples from 14 different sampling sites on the human body and finally, we assessed the impact of age and sex on lipidome variability in 104 healthy subjects. We found sebaceous lipids to constitute an abundant component of the SC lipidome as they diffuse into the topmost SC layers forming a gradient. Lipidomic variability with respect to sampling depth, site and subject is considerable, and mainly accredited to sebaceous lipids, while stratum corneum lipids vary less. This stresses the importance of sampling design and the role of sebaceous lipids in skin studies.

SUPER-FOCUS: a tool for agile functional analysis of shotgun metagenomic data.

PubMed

Silva, Genivaldo Gueiros Z; Green, Kevin T; Dutilh, Bas E; Edwards, Robert A

2016-02-01

Analyzing the functional profile of a microbial community from unannotated shotgun sequencing reads is one of the important goals in metagenomics. Functional profiling has valuable applications in biological research because it identifies the abundances of the functional genes of the organisms present in the original sample, answering the question what they can do. Currently, available tools do not scale well with increasing data volumes, which is important because both the number and lengths of the reads produced by sequencing platforms keep increasing. Here, we introduce SUPER-FOCUS, SUbsystems Profile by databasE Reduction using FOCUS, an agile homology-based approach using a reduced reference database to report the subsystems present in metagenomic datasets and profile their abundances. SUPER-FOCUS was tested with over 70 real metagenomes, the results showing that it accurately predicts the subsystems present in the profiled microbial communities, and is up to 1000 times faster than other tools. SUPER-FOCUS was implemented in Python, and its source code and the tool website are freely available at https://edwards.sdsu.edu/SUPERFOCUS. redwards@mail.sdsu.edu Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press.

[Left ventricular projectile migration after an accidental close-range gunshot wound].

PubMed

Driessen, A; Tjardes, T; Eikermann, C; Trojan, S; Fröhlich, M; Grimaldi, G; Kosse, N

2016-07-01

We report the case of a 24-year-old female after sustaining a shotgun wound in the left upper extremity and chest. Initial emergency diagnostics revealed numerous shotgun pellets scattered throughout the left-side soft tissue, chest and upper lung lobe with one pellet having migrated into the left ventricle of the heart.Due to the devastating injury pattern, gunshot wounds are interdisciplinarily challenging and should include extended initial diagnostics, such as contrast agent CT. The potential toxicity of elevated lead blood levels have to be taken into further account.

The single-species metagenome: subtyping Staphylococcus aureus core genome sequences from shotgun metagenomic data

PubMed Central

Li, Ben; Petit III, Robert A.; Qin, Zhaohui S.; Darrow, Lyndsey

2016-01-01

In this study we developed a genome-based method for detecting Staphylococcus aureus subtypes from metagenome shotgun sequence data. We used a binomial mixture model and the coverage counts at >100,000 known S. aureus SNP (single nucleotide polymorphism) sites derived from prior comparative genomic analysis to estimate the proportion of 40 subtypes in metagenome samples. We were able to obtain >87% sensitivity and >94% specificity at 0.025X coverage for S. aureus. We found that 321 and 149 metagenome samples from the Human Microbiome Project and metaSUB analysis of the New York City subway, respectively, contained S. aureus at genome coverage >0.025. In both projects, CC8 and CC30 were the most common S. aureus clonal complexes encountered. We found evidence that the subtype composition at different body sites of the same individual were more similar than random sampling and more limited evidence that certain body sites were enriched for particular subtypes. One surprising finding was the apparent high frequency of CC398, a lineage often associated with livestock, in samples from the tongue dorsum. Epidemiologic analysis of the HMP subject population suggested that high BMI (body mass index) and health insurance are possibly associated with S. aureus carriage but there was limited power to identify factors linked to carriage of even the most common subtype. In the NYC subway data, we found a small signal of geographic distance affecting subtype clustering but other unknown factors influence taxonomic distribution of the species around the city. PMID:27781166

Quantitative Proteomic Analysis of Differentially Expressed Protein Profiles Involved in Pancreatic Ductal Adenocarcinoma

PubMed Central

Kuo, Kung-Kai; Kuo, Chao-Jen; Chiu, Chiang-Yen; Liang, Shih-Shin; Huang, Chun-Hao; Chi, Shu-Wen; Tsai, Kun-Bow; Chen, Chiao-Yun; Hsi, Edward; Cheng, Kuang-Hung; Chiou, Shyh-Horng

2016-01-01

Objectives The aim of this study was to identify differentially expressed proteins among various stages of pancreatic ductal adenocarcinoma (PDAC) by shotgun proteomics using nano-liquid chromatography coupled tandem mass spectrometry and stable isotope dimethyl labeling. Methods Differentially expressed proteins were identified and compared based on the mass spectral differences of their isotope-labeled peptide fragments generated from protease digestion. Results Our quantitative proteomic analysis of the differentially expressed proteins with stable isotope (deuterium/hydrogen ratio, ≥2) identified a total of 353 proteins, with at least 5 protein biomarker proteins that were significantly differentially expressed between cancer and normal mice by at least a 2-fold alteration. These 5 protein biomarker candidates include α-enolase, α-catenin, 14-3-3 β, VDAC1, and calmodulin with high confidence levels. The expression levels were also found to be in agreement with those examined by Western blot and histochemical staining. Conclusions The systematic decrease or increase of these identified marker proteins may potentially reflect the morphological aberrations and diseased stages of pancreas carcinoma throughout progressive developments leading to PDAC. The results would form a firm foundation for future work concerning validation and clinical translation of some identified biomarkers into targeted diagnosis and therapy for various stages of PDAC. PMID:26262590

An Integrated Proteomics and Bioinformatics Approach Reveals the Anti-inflammatory Mechanism of Carnosic Acid

PubMed Central

Wang, Li-Chao; Wei, Wen-Hui; Zhang, Xiao-Wen; Liu, Dan; Zeng, Ke-Wu; Tu, Peng-Fei

2018-01-01

Drastic macrophages activation triggered by exogenous infection or endogenous stresses is thought to be implicated in the pathogenesis of various inflammatory diseases. Carnosic acid (CA), a natural phenolic diterpene extracted from Salvia officinalis plant, has been reported to possess anti-inflammatory activity. However, its role in macrophages activation as well as potential molecular mechanism is largely unexplored. In the current study, we sought to elucidate the anti-inflammatory property of CA using an integrated approach based on unbiased proteomics and bioinformatics analysis. CA significantly inhibited the robust increase of nitric oxide and TNF-α, downregulated COX2 protein expression, and lowered the transcriptional level of inflammatory genes including Nos2, Tnfα, Cox2, and Mcp1 in LPS-stimulated RAW264.7 cells, a murine model of peritoneal macrophage cell line. The LC-MS/MS-based shotgun proteomics analysis showed CA negatively regulated 217 LPS-elicited proteins which were involved in multiple inflammatory processes including MAPK, nuclear factor (NF)-κB, and FoxO signaling pathways. A further molecular biology analysis revealed that CA effectually inactivated IKKβ/IκB-α/NF-κB, ERK/JNK/p38 MAPKs, and FoxO1/3 signaling pathways. Collectively, our findings demonstrated the role of CA in regulating inflammation response and provide some insights into the proteomics-guided pharmacological mechanism study of natural products. PMID:29713284

Cost-Effective Sequencing of Full-Length cDNA Clones Powered by a De Novo-Reference Hybrid Assembly

PubMed Central

Sugano, Sumio; Morishita, Shinichi; Suzuki, Yutaka

2010-01-01

Background Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded. Methodology We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence ∼800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species. Conclusions The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only ∼US$3 per clone, demonstrating a significant advantage over previous approaches. PMID:20479877

Genomics approach to the environmental community of microorganisms

NASA Astrophysics Data System (ADS)

Kawarabayasi, Y.; Maruyama, A.

2004-12-01

It was indicated by microscopic observation or comparison of 16S rDNA sequence that many extremophiles were surviving in many hydrothermal environments. But it is generally said that over 99% of total microbes are now uncultivable. Thus, we planned to identify uncultivable microbes through direct sequencing of environmental DNA. At first, shotgun plasmid libraries were directly constructed with the DNA molecules prepared from mixed microbes collected from low-temperature hydrothermal water at RM24 in the Southern East Pacific Rise (S-EPR). It was shown that the sequences of some number of clones indicated the similar feature to the intron in eukaryote or tandem repetitive sequence identified in some human familiar diseases. The results indicated that many microorganisms with eukaryotic feature were dominant in low temperature water of S-EPR. Secondly, shotgun plasmid libraries were constructed from the environmental DNA prepared from Beppu hot springs. The ORFs were easily identified all clones determined entire sequence. Thus it can be said that hot springs is good resources for searching novel genes. At last, the mixed microbes isolated from Suiyo seamount were used for construction of shotgun library. The clones in this library contained the ORFs. From some clones in hot spring and Suiyo sample, aminoacyl-tRNA synthatase, which is generally present in all organisms, was isolated by similarity. The phylogenetic analysis of aminoacyl-tRNA synthetase identified indicated that novel and unidentified microorganisms should be present in hot spring or Suiyo seamount. The novel genes identified from Suiyo seamount were also utilized for expression in E. coli. Some gene products were successfully obtained from the E. coli cells as soluble proteins. Some protein indicated the thermostability up to 70_E#8249;C, meaning that the original host cell of this gene should be stable up to the same temperature. Our work indicates that environmental genomics, including the direct cloning, sequencing of environmental DNA and expression of gene identified, is powerful approach to collect novel uncultivable microbes or novel active genes.

Applicability of a high-throughput shotgun plasma protein screening approach in understanding maternal biological pathways relevant to infant birth weight outcome.

PubMed

Kumarathasan, P; Vincent, R; Das, D; Mohottalage, S; Blais, E; Blank, K; Karthikeyan, S; Vuong, N Q; Arbuckle, T E; Fraser, W D

2014-04-04

There are reports linking maternal nutritional status, smoking and environmental chemical exposures to adverse pregnancy outcomes. However, biological bases for association between some of these factors and birth outcomes are yet to be established. The objective of this preliminary work is to test the capability of a new high-throughput shotgun plasma proteomic screening in identifying maternal changes relevant to pregnancy outcome. A subset of third trimester plasma samples (N=12) associated with normal and low-birth weight infants were fractionated, tryptic-digested and analyzed for global proteomic changes using a MALDI-TOF-TOF-MS methodology. Mass spectral data were mined for candidate biomarkers using bioinformatic and statistical tools. Maternal plasma profiles of cytokines (e.g. IL8, TNF-α), chemokines (e.g. MCP-1) and cardiovascular endpoints (e.g. ET-1, MMP-9) were analyzed by a targeted approach using multiplex protein array and HPLC-Fluorescence methods. Target and global plasma proteomic markers were used to identify protein interaction networks and maternal biological pathways relevant to low infant birth weight. Our results exhibited the potential to discriminate specific maternal physiologies relevant to risk of adverse birth outcomes. This proteomic approach can be valuable in understanding the impacts of maternal factors such as environmental contaminant exposures and nutrition on birth outcomes in future work. We demonstrate here the fitness of mass spectrometry-based shot-gun proteomics for surveillance of biological changes in mothers, and for adverse pathway analysis in combination with target biomarker information. This approach has potential for enabling early detection of mothers at risk for low infant birth weight and preterm birth, and thus early intervention for mitigation and prevention of adverse pregnancy outcomes. This article is part of a Special Issue entitled: Can Proteomics Fill the Gap Between Genomics and Phenotypes? Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Proteomic analysis of Fasciola hepatica excretory and secretory products (FhESPs) involved in interacting with host PBMCs and cytokines by shotgun LC-MS/MS.

PubMed

Liu, Qing; Huang, Si-Yang; Yue, Dong-Mei; Wang, Jin-Lei; Wang, Yujian; Li, Xiangrui; Zhu, Xing-Quan

2017-02-01

Fasciola hepatica is a helminth parasite with a worldwide distribution, which can cause chronic liver disease, fasciolosis, leading to economic losses in the livestock and public health in many countries. Control is mostly reliant on the use of drugs, and as a result, drug resistance has now emerged. The identification of F. hepatica genes involved in interaction between the parasite and host immune system is utmost important to elucidate the evasion mechanisms of the parasite and develop more effective strategies against fasciolosis. In this study, we aimed to identify molecules in F. hepatica excretory and secretory products (FhESPs) interacting with the host peripheral blood mononuclear cells (PBMCs), Th1-like cytokines (IL2 and IFN-γ), and Th17-like cytokines (IL17) by Co-IP combined with tandem mass spectrometry. The results showed that 14, 16, and 9 proteins in FhESPs could bind with IL2, IL17, and IFN-γ, respectively, which indicated that adult F. hepatica may evade the host immune responses through directly interplaying with cytokines. In addition, nine proteins in FhESPs could adhere to PBMCs. Our findings provided potential targets as immuno-regulators, and will be helpful to elucidate the molecular basis of host-parasite interactions and search for new potential proteins as vaccine and drug target candidates.

Quantitative proteomic analysis of cabernet sauvignon grape cells exposed to thermal stresses reveals alterations in sugar and phenylpropanoid metabolism.

PubMed

George, Iniga S; Pascovici, Dana; Mirzaei, Mehdi; Haynes, Paul A

2015-09-01

Grapes (Vitis vinifera) are a valuable fruit crop and wine production is a major industry. Global warming and expanded range of cultivation will expose grapes to more temperature stresses in future. Our study investigated protein level responses to abiotic stresses, with particular reference to proteomic changes induced by the impact of four different temperature stress regimes, including both hot and cold temperatures, on cultured grape cells. Cabernet Sauvignon cell suspension cultures grown at 26°C were subjected to 14 h of exposure to 34 and 42°C for heat stress, and 18 and 10°C for cold stress. Cells from the five temperatures were harvested in biological triplicates and label-free quantitative shotgun proteomic analysis was performed. A total of 2042 non-redundant proteins were identified from the five temperature points. Fifty-five proteins were only detected in extreme heat stress conditions (42°C) and 53 proteins were only detected at extreme cold stress conditions (10°C). Gene Ontology (GO) annotations of differentially expressed proteins provided insights into the metabolic pathways that are involved in temperature stress in grape cells. Sugar metabolism displayed switching between alternative and classical pathways during temperature stresses. Additionally, nine proteins involved in the phenylpropanoid pathway were greatly increased in abundance at extreme cold stress, and were thus found to be cold-responsive proteins. All MS data have been deposited in the ProteomeXchange with identifier PXD000977 (http://proteomecentral.proteomexchange.org/dataset/PXD000977). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomic analysis reveals metabolic and regulatory systems involved in the syntrophic and axenic lifestyle of Syntrophomonas wolfei

DOE PAGES

Sieber, Jessica R.; Crable, Bryan R.; Sheik, Cody S.; ...

2015-02-11

We report that microbial syntrophy is a vital metabolic interaction necessary for the complete oxidation of organic biomass to methane in all-anaerobic ecosystems. However, this process is thermodynamically constrained and represents an ecosystem-level metabolic bottleneck. To gain insight into the physiology of this process, a shotgun proteomics approach was used to quantify the protein landscape of the model syntrophic metabolizer, Syntrophomonas wolfei, grown axenically and syntrophically with Methanospirillum hungatei. Remarkably, the abundance of most proteins as represented by normalized spectral abundance factor (NSAF) value changed very little between the pure and coculture growth conditions. Among the most abundant proteins detectedmore » were GroEL and GroES chaperonins, a small heat shock protein, and proteins involved in electron transfer, beta-oxidation, and ATP synthesis. Several putative energy conservation enzyme systems that utilize NADH and ferredoxin were present. The abundance of an EtfAB2 and the membrane-bound iron-sulfur oxidoreductase (Swol_0698 gene product) delineated a potential conduit for electron transfer between acyl-CoA dehydrogenases and membrane redox carriers. Proteins detected only when S. wolfei was grown with M. hungatei included a zinc-dependent dehydrogenase with a GroES domain, whose gene is present in genomes in many organisms capable of syntrophy, and transcriptional regulators responsive to environmental stimuli or the physiological status of the cell. In conclusion, the proteomic analysis revealed an emphasis on macromolecular stability and energy metabolism by S. wolfei and presence of regulatory mechanisms responsive to external stimuli and cellular physiological status.« less

Proteomic analysis reveals metabolic and regulatory systems involved in the syntrophic and axenic lifestyle of Syntrophomonas wolfei

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

Sieber, Jessica R.; Crable, Bryan R.; Sheik, Cody S.

We report that microbial syntrophy is a vital metabolic interaction necessary for the complete oxidation of organic biomass to methane in all-anaerobic ecosystems. However, this process is thermodynamically constrained and represents an ecosystem-level metabolic bottleneck. To gain insight into the physiology of this process, a shotgun proteomics approach was used to quantify the protein landscape of the model syntrophic metabolizer, Syntrophomonas wolfei, grown axenically and syntrophically with Methanospirillum hungatei. Remarkably, the abundance of most proteins as represented by normalized spectral abundance factor (NSAF) value changed very little between the pure and coculture growth conditions. Among the most abundant proteins detectedmore » were GroEL and GroES chaperonins, a small heat shock protein, and proteins involved in electron transfer, beta-oxidation, and ATP synthesis. Several putative energy conservation enzyme systems that utilize NADH and ferredoxin were present. The abundance of an EtfAB2 and the membrane-bound iron-sulfur oxidoreductase (Swol_0698 gene product) delineated a potential conduit for electron transfer between acyl-CoA dehydrogenases and membrane redox carriers. Proteins detected only when S. wolfei was grown with M. hungatei included a zinc-dependent dehydrogenase with a GroES domain, whose gene is present in genomes in many organisms capable of syntrophy, and transcriptional regulators responsive to environmental stimuli or the physiological status of the cell. In conclusion, the proteomic analysis revealed an emphasis on macromolecular stability and energy metabolism by S. wolfei and presence of regulatory mechanisms responsive to external stimuli and cellular physiological status.« less

Shotgun proteomics reveals specific modulated protein patterns in tears of patients with primary open angle glaucoma naïve to therapy.

PubMed

Pieragostino, Damiana; Agnifili, Luca; Fasanella, Vincenzo; D'Aguanno, Simona; Mastropasqua, Rodolfo; Di Ilio, Carmine; Sacchetta, Paolo; Urbani, Andrea; Del Boccio, Piero

2013-06-01

Primary open angle glaucoma (POAG) is one of the main causes of irreversible blindness worldwide. The pathogenesis of POAG is still unclear. Alteration and sclerosis of trabecular meshwork with changes in aqueous humor molecular composition seem to play the key role. Increased intraocular pressure is widely known to be the main risk factor for the onset and progression of the disease. Unfortunately, the early diagnosis of POAG still remains the main challenge. In order to provide insight into the patho-physiology of glaucoma, here we report a shotgun proteomics approach to tears of patients with POAG naïve to therapy. Our proteomics results showed 27 differential tear proteins in POAG vs. CTRL comparison (25 up regulated proteins in the POAG group and two unique proteins in the CTRL group), 16 of which were associated with inflammatory response, free radical scavenging, cell-to-cell signaling and interaction. Overall the protein modulation shown in POAG tears proves the involvement of biochemical networks linked to inflammation. Among all regulated proteins, a sub-group of 12 up-regulated proteins in naïve POAG patients were found to be down-regulated in medically controlled POAG patients treated with prostanoid analogues (PGA), as reported in our previous work (i.e., lipocalin-1, lysozyme C, lactotransferrin, proline-rich-protein 4, prolactin-inducible protein, zinc-alpha-2-glycoprotein, polymeric immunoglobulin receptor, cystatin S, Ig kappa chain C region, Ig alpha-2 chain C region, immunoglobulin J chain, Ig alpha-1 chain C region). In summary, our findings indicate that the POAG tears protein expression is a mixture of increased inflammatory proteins that could be potential biomarkers of the disease, and their regulation may be involved in the mechanism by which PGA are able to decrease the intraocular pressure in glaucoma patients.

[Wound ballistics of extremely short-range shotguns].

PubMed

Rabl, W; Eller, W

1993-01-01

A 45-year-old man died from a homicidal shotgun wound of the neck. The autopsy showed that the plastic shell retaining cup had entered the body after the mass of the shot; the dispersion of the individual pellets was about 12 cm already. The perpetrator had used a conventional signal pistol equipped with a hand-made steel barrel, gauge 12, length 20 cm, and ammunition with 3 mm pellets (shot no. 5). By means of experimental shots with a similar weapon it could be proved that the entire shot including the plastic shell retaining cup can penetrate the skin at a muzzle-victim range of less than 2 m.

Library Construction from Subnanogram DNA for Pelagic Sea Water and Deep-Sea Sediments

PubMed Central

Hirai, Miho; Nishi, Shinro; Tsuda, Miwako; Sunamura, Michinari; Takaki, Yoshihiro; Nunoura, Takuro

2017-01-01

Shotgun metagenomics is a low biased technology for assessing environmental microbial diversity and function. However, the requirement for a sufficient amount of DNA and the contamination of inhibitors in environmental DNA leads to difficulties in constructing a shotgun metagenomic library. We herein examined metagenomic library construction from subnanogram amounts of input environmental DNA from subarctic surface water and deep-sea sediments using two library construction kits: the KAPA Hyper Prep Kit and Nextera XT DNA Library Preparation Kit, with several modifications. The influence of chemical contaminants associated with these environmental DNA samples on library construction was also investigated. Overall, shotgun metagenomic libraries were constructed from 1 pg to 1 ng of input DNA using both kits without harsh library microbial contamination. However, the libraries constructed from 1 pg of input DNA exhibited larger biases in GC contents, k-mers, or small subunit (SSU) rRNA gene compositions than those constructed from 10 pg to 1 ng DNA. The lower limit of input DNA for low biased library construction in this study was 10 pg. Moreover, we revealed that technology-dependent biases (physical fragmentation and linker ligation vs. tagmentation) were larger than those due to the amount of input DNA. PMID:29187708

Comparison of three-dimensional particle tracking and sizing using plenoptic imaging and digital in-line holography.

PubMed

Hall, Elise M; Thurow, Brian S; Guildenbecher, Daniel R

2016-08-10

Digital in-line holography (DIH) and plenoptic photography are two techniques for single-shot, volumetric measurement of 3D particle fields. Here we present a comparison of the two methods by applying plenoptic imaging to experimental configurations that have been previously investigated with DIH. These experiments include the tracking of secondary droplets from the impact of a water drop on a thin film of water and tracking of pellets from a shotgun. Both plenoptic imaging and DIH successfully quantify the 3D nature of these particle fields. This includes measurement of the 3D particle position, individual particle sizes, and three-component velocity vectors. For the initial processing methods presented here, both techniques give out-of-plane positional accuracy of approximately 1-2 particle diameters. For a fixed image sensor, digital holography achieves higher effective in-plane spatial resolutions. However, collimated and coherent illumination makes holography susceptible to image distortion through index of refraction gradients, as demonstrated in the shotgun experiments. In contrast, plenoptic imaging allows for a simpler experimental configuration and, due to the use of diffuse, white-light illumination, plenoptic imaging is less susceptible to image distortion in the shotgun experiments.

How to talk about protein‐level false discovery rates in shotgun proteomics

PubMed Central

The, Matthew; Tasnim, Ayesha

2016-01-01

A frequently sought output from a shotgun proteomics experiment is a list of proteins that we believe to have been present in the analyzed sample before proteolytic digestion. The standard technique to control for errors in such lists is to enforce a preset threshold for the false discovery rate (FDR). Many consider protein‐level FDRs a difficult and vague concept, as the measurement entities, spectra, are manifestations of peptides and not proteins. Here, we argue that this confusion is unnecessary and provide a framework on how to think about protein‐level FDRs, starting from its basic principle: the null hypothesis. Specifically, we point out that two competing null hypotheses are used concurrently in today's protein inference methods, which has gone unnoticed by many. Using simulations of a shotgun proteomics experiment, we show how confusing one null hypothesis for the other can lead to serious discrepancies in the FDR. Furthermore, we demonstrate how the same simulations can be used to verify FDR estimates of protein inference methods. In particular, we show that, for a simple protein inference method, decoy models can be used to accurately estimate protein‐level FDRs for both competing null hypotheses. PMID:27503675

Shotgun glycomics of pig lung identifies natural endogenous receptors for influenza viruses.

PubMed

Byrd-Leotis, Lauren; Liu, Renpeng; Bradley, Konrad C; Lasanajak, Yi; Cummings, Sandra F; Song, Xuezheng; Heimburg-Molinaro, Jamie; Galloway, Summer E; Culhane, Marie R; Smith, David F; Steinhauer, David A; Cummings, Richard D

2014-06-03

Influenza viruses bind to host cell surface glycans containing terminal sialic acids, but as studies on influenza binding become more sophisticated, it is becoming evident that although sialic acid may be necessary, it is not sufficient for productive binding. To better define endogenous glycans that serve as viral receptors, we have explored glycan recognition in the pig lung, because influenza is broadly disseminated in swine, and swine have been postulated as an intermediary host for the emergence of pandemic strains. For these studies, we used the technology of "shotgun glycomics" to identify natural receptor glycans. The total released N- and O-glycans from pig lung glycoproteins and glycolipid-derived glycans were fluorescently tagged and separated by multidimensional HPLC, and individual glycans were covalently printed to generate pig lung shotgun glycan microarrays. All viruses tested interacted with one or more sialylated N-glycans but not O-glycans or glycolipid-derived glycans, and each virus demonstrated novel and unexpected differences in endogenous N-glycan recognition. The results illustrate the repertoire of specific, endogenous N-glycans of pig lung glycoproteins for virus recognition and offer a new direction for studying endogenous glycan functions in viral pathogenesis.

Quantitative proteomic analysis of intact plastids.

PubMed

Shiraya, Takeshi; Kaneko, Kentaro; Mitsui, Toshiaki

2014-01-01

Plastids are specialized cell organelles in plant cells that are differentiated into various forms including chloroplasts, chromoplasts, and amyloplasts, and fulfill important functions in maintaining the overall cell metabolism and sensing environmental factors such as sunlight. It is therefore important to grasp the mechanisms of differentiation and functional changes of plastids in order to enhance the understanding of vegetality. In this chapter, details of a method for the extraction of intact plastids that makes analysis possible while maintaining the plastid functions are provided; in addition, a quantitative shotgun method for analyzing the composition and changes in the content of proteins in plastids as a result of environmental impacts is described.

Applications of mass spectrometry for quantitative protein analysis in formalin-fixed paraffin-embedded tissues

PubMed Central

Steiner, Carine; Ducret, Axel; Tille, Jean-Christophe; Thomas, Marlene; McKee, Thomas A; Rubbia-Brandt, Laura A; Scherl, Alexander; Lescuyer, Pierre; Cutler, Paul

2014-01-01

Proteomic analysis of tissues has advanced in recent years as instruments and methodologies have evolved. The ability to retrieve peptides from formalin-fixed paraffin-embedded tissues followed by shotgun or targeted proteomic analysis is offering new opportunities in biomedical research. In particular, access to large collections of clinically annotated samples should enable the detailed analysis of pathologically relevant tissues in a manner previously considered unfeasible. In this paper, we review the current status of proteomic analysis of formalin-fixed paraffin-embedded tissues with a particular focus on targeted approaches and the potential for this technique to be used in clinical research and clinical diagnosis. We also discuss the limitations and perspectives of the technique, particularly with regard to application in clinical diagnosis and drug discovery. PMID:24339433

Comprehensive and quantitative profiling of lipid species in human milk, cow milk and a phospholipid-enriched milk formula by GC and MS/MSALL.

PubMed

Sokol, Elena; Ulven, Trond; Færgeman, Nils J; Ejsing, Christer S

2015-06-01

Here we present a workflow for in-depth analysis of milk lipids that combines gas chromatography (GC) for fatty acid (FA) profiling and a shotgun lipidomics routine termed MS/MS ALL for structural characterization of molecular lipid species. To evaluate the performance of the workflow we performed a comparative lipid analysis of human milk, cow milk, and Lacprodan® PL-20, a phospholipid-enriched milk protein concentrate for infant formula. The GC analysis showed that human milk and Lacprodan have a similar FA profile with higher levels of unsaturated FAs as compared to cow milk. In-depth lipidomic analysis by MS/MS ALL revealed that each type of milk sample comprised distinct composition of molecular lipid species. Lipid class composition showed that the human and cow milk contain a higher proportion of triacylglycerols (TAGs) as compared to Lacprodan. Notably, the MS/MS ALL analysis demonstrated that the similar FA profile of human milk and Lacprodan determined by GC analysis is attributed to the composition of individual TAG species in human milk and glycerophospholipid species in Lacprodan. Moreover, the analysis of TAG molecules in Lacprodan and cow milk showed a high proportion of short-chain FAs that could not be monitored by GC analysis. The results presented here show that complementary GC and MS/MS ALL analysis is a powerful approach for characterization of molecular lipid species in milk and milk products. : Milk lipid analysis is routinely performed using gas chromatography. This method reports the total fatty acid composition of all milk lipids, but provides no structural or quantitative information about individual lipid molecules in milk or milk products. Here we present a workflow that integrates gas chromatography for fatty acid profiling and a shotgun lipidomics routine termed MS/MS ALL for structural analysis and quantification of molecular lipid species. We demonstrate the efficacy of this complementary workflow by a comparative analysis of molecular lipid species in human milk, cow milk, and a milk-based supplement used for infant formula.

Comprehensive and quantitative profiling of lipid species in human milk, cow milk and a phospholipid-enriched milk formula by GC and MS/MSALL

PubMed Central

Sokol, Elena; Ulven, Trond; Færgeman, Nils J; Ejsing, Christer S

2015-01-01

Here we present a workflow for in-depth analysis of milk lipids that combines gas chromatography (GC) for fatty acid (FA) profiling and a shotgun lipidomics routine termed MS/MSALL for structural characterization of molecular lipid species. To evaluate the performance of the workflow we performed a comparative lipid analysis of human milk, cow milk, and Lacprodan® PL-20, a phospholipid-enriched milk protein concentrate for infant formula. The GC analysis showed that human milk and Lacprodan have a similar FA profile with higher levels of unsaturated FAs as compared to cow milk. In-depth lipidomic analysis by MS/MSALL revealed that each type of milk sample comprised distinct composition of molecular lipid species. Lipid class composition showed that the human and cow milk contain a higher proportion of triacylglycerols (TAGs) as compared to Lacprodan. Notably, the MS/MSALL analysis demonstrated that the similar FA profile of human milk and Lacprodan determined by GC analysis is attributed to the composition of individual TAG species in human milk and glycerophospholipid species in Lacprodan. Moreover, the analysis of TAG molecules in Lacprodan and cow milk showed a high proportion of short-chain FAs that could not be monitored by GC analysis. The results presented here show that complementary GC and MS/MSALL analysis is a powerful approach for characterization of molecular lipid species in milk and milk products. Practical applications : Milk lipid analysis is routinely performed using gas chromatography. This method reports the total fatty acid composition of all milk lipids, but provides no structural or quantitative information about individual lipid molecules in milk or milk products. Here we present a workflow that integrates gas chromatography for fatty acid profiling and a shotgun lipidomics routine termed MS/MSALL for structural analysis and quantification of molecular lipid species. We demonstrate the efficacy of this complementary workflow by a comparative analysis of molecular lipid species in human milk, cow milk, and a milk-based supplement used for infant formula. PMID:26089741

Genome Improvement at JGI-HAGSC

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

Grimwood, Jane; Schmutz, Jeremy J.; Myers, Richard M.

Since the completion of the sequencing of the human genome, the Joint Genome Institute (JGI) has rapidly expanded its scientific goals in several DOE mission-relevant areas. At the JGI-HAGSC, we have kept pace with this rapid expansion of projects with our focus on assessing, assembling, improving and finishing eukaryotic whole genome shotgun (WGS) projects for which the shotgun sequence is generated at the Production Genomic Facility (JGI-PGF). We follow this by combining the draft WGS with genomic resources generated at JGI-HAGSC or in collaborator laboratories (including BAC end sequences, genetic maps and FLcDNA sequences) to produce an improved draft sequence.more » For eukaryotic genomes important to the DOE mission, we then add further information from directed experiments to produce reference genomic sequences that are publicly available for any scientific researcher. Also, we have continued our program for producing BAC-based finished sequence, both for adding information to JGI genome projects and for small BAC-based sequencing projects proposed through any of the JGI sequencing programs. We have now built our computational expertise in WGS assembly and analysis and have moved eukaryotic genome assembly from the JGI-PGF to JGI-HAGSC. We have concentrated our assembly development work on large plant genomes and complex fungal and algal genomes.« less

A survey of computational methods and error rate estimation procedures for peptide and protein identification in shotgun proteomics

PubMed Central

Nesvizhskii, Alexey I.

2010-01-01

This manuscript provides a comprehensive review of the peptide and protein identification process using tandem mass spectrometry (MS/MS) data generated in shotgun proteomic experiments. The commonly used methods for assigning peptide sequences to MS/MS spectra are critically discussed and compared, from basic strategies to advanced multi-stage approaches. A particular attention is paid to the problem of false-positive identifications. Existing statistical approaches for assessing the significance of peptide to spectrum matches are surveyed, ranging from single-spectrum approaches such as expectation values to global error rate estimation procedures such as false discovery rates and posterior probabilities. The importance of using auxiliary discriminant information (mass accuracy, peptide separation coordinates, digestion properties, and etc.) is discussed, and advanced computational approaches for joint modeling of multiple sources of information are presented. This review also includes a detailed analysis of the issues affecting the interpretation of data at the protein level, including the amplification of error rates when going from peptide to protein level, and the ambiguities in inferring the identifies of sample proteins in the presence of shared peptides. Commonly used methods for computing protein-level confidence scores are discussed in detail. The review concludes with a discussion of several outstanding computational issues. PMID:20816881

Mass spectrometry-based targeted quantitative proteomics: achieving sensitive and reproducible detection of proteins.

PubMed

Boja, Emily S; Rodriguez, Henry

2012-04-01

Traditional shotgun proteomics used to detect a mixture of hundreds to thousands of proteins through mass spectrometric analysis, has been the standard approach in research to profile protein content in a biological sample which could lead to the discovery of new (and all) protein candidates with diagnostic, prognostic, and therapeutic values. In practice, this approach requires significant resources and time, and does not necessarily represent the goal of the researcher who would rather study a subset of such discovered proteins (including their variations or posttranslational modifications) under different biological conditions. In this context, targeted proteomics is playing an increasingly important role in the accurate measurement of protein targets in biological samples in the hope of elucidating the molecular mechanism of cellular function via the understanding of intricate protein networks and pathways. One such (targeted) approach, selected reaction monitoring (or multiple reaction monitoring) mass spectrometry (MRM-MS), offers the capability of measuring multiple proteins with higher sensitivity and throughput than shotgun proteomics. Developing and validating MRM-MS-based assays, however, is an extensive and iterative process, requiring a coordinated and collaborative effort by the scientific community through the sharing of publicly accessible data and datasets, bioinformatic tools, standard operating procedures, and well characterized reagents. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human.

PubMed

Li, Caixia; Tan, Xing Fei; Lim, Teck Kwang; Lin, Qingsong; Gong, Zhiyuan

2016-04-13

Omic approaches have been increasingly used in the zebrafish model for holistic understanding of molecular events and mechanisms of tissue functions. However, plasma is rarely used for omic profiling because of the technical challenges in collecting sufficient blood. In this study, we employed two mass spectrometric (MS) approaches for a comprehensive characterization of zebrafish plasma proteome, i.e. conventional shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) for an overview study and quantitative SWATH (Sequential Window Acquisition of all THeoretical fragment-ion spectra) for comparison between genders. 959 proteins were identified in the shotgun profiling with estimated concentrations spanning almost five orders of magnitudes. Other than the presence of a few highly abundant female egg yolk precursor proteins (vitellogenins), the proteomic profiles of male and female plasmas were very similar in both number and abundance and there were basically no other highly gender-biased proteins. The types of plasma proteins based on IPA (Ingenuity Pathway Analysis) classification and tissue sources of production were also very similar. Furthermore, the zebrafish plasma proteome shares significant similarities with human plasma proteome, in particular in top abundant proteins including apolipoproteins and complements. Thus, the current study provided a valuable dataset for future evaluation of plasma proteins in zebrafish.

Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human

PubMed Central

Li, Caixia; Tan, Xing Fei; Lim, Teck Kwang; Lin, Qingsong; Gong, Zhiyuan

2016-01-01

Omic approaches have been increasingly used in the zebrafish model for holistic understanding of molecular events and mechanisms of tissue functions. However, plasma is rarely used for omic profiling because of the technical challenges in collecting sufficient blood. In this study, we employed two mass spectrometric (MS) approaches for a comprehensive characterization of zebrafish plasma proteome, i.e. conventional shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) for an overview study and quantitative SWATH (Sequential Window Acquisition of all THeoretical fragment-ion spectra) for comparison between genders. 959 proteins were identified in the shotgun profiling with estimated concentrations spanning almost five orders of magnitudes. Other than the presence of a few highly abundant female egg yolk precursor proteins (vitellogenins), the proteomic profiles of male and female plasmas were very similar in both number and abundance and there were basically no other highly gender-biased proteins. The types of plasma proteins based on IPA (Ingenuity Pathway Analysis) classification and tissue sources of production were also very similar. Furthermore, the zebrafish plasma proteome shares significant similarities with human plasma proteome, in particular in top abundant proteins including apolipoproteins and complements. Thus, the current study provided a valuable dataset for future evaluation of plasma proteins in zebrafish. PMID:27071722

Cloud CPFP: a shotgun proteomics data analysis pipeline using cloud and high performance computing.

PubMed

Trudgian, David C; Mirzaei, Hamid

2012-12-07

We have extended the functionality of the Central Proteomics Facilities Pipeline (CPFP) to allow use of remote cloud and high performance computing (HPC) resources for shotgun proteomics data processing. CPFP has been modified to include modular local and remote scheduling for data processing jobs. The pipeline can now be run on a single PC or server, a local cluster, a remote HPC cluster, and/or the Amazon Web Services (AWS) cloud. We provide public images that allow easy deployment of CPFP in its entirety in the AWS cloud. This significantly reduces the effort necessary to use the software, and allows proteomics laboratories to pay for compute time ad hoc, rather than obtaining and maintaining expensive local server clusters. Alternatively the Amazon cloud can be used to increase the throughput of a local installation of CPFP as necessary. We demonstrate that cloud CPFP allows users to process data at higher speed than local installations but with similar cost and lower staff requirements. In addition to the computational improvements, the web interface to CPFP is simplified, and other functionalities are enhanced. The software is under active development at two leading institutions and continues to be released under an open-source license at http://cpfp.sourceforge.net.

[The ultrasonic diagnosis of jaundice. 199 cases (author's transl)].

PubMed

Weill, F; Marmier, A; Paronneau, P; Zeltner, F; Charton, M N

1978-11-25

Thank to a thorough ultrasonographic analysis of biliary tree ("shotgun sign"), liver and pancreas, a positive diagnosis of obstruction was carried out in 92% of cases. The success rate in diagnosis of level of obstruction was also 92%. Aetiologic diagnosis was successful in 61% of cases only (almost 100% in jaundices of pancreatic origine). No false positive diagnosis of obstruction was made in non-obstructive jaundice. This enabled to carry out instrumental cholangiography (i.e. "skinny" needle percutaneous cholangiography, and ERC) only in case of clinical, biological and sonographic discrepancies, or in hilar obstructions.

MassSieve: Panning MS/MS peptide data for proteins

PubMed Central

Slotta, Douglas J.; McFarland, Melinda A.; Markey, Sanford P.

2010-01-01

We present MassSieve, a Java-based platform for visualization and parsimony analysis of single and comparative LC-MS/MS database search engine results. The success of mass spectrometric peptide sequence assignment algorithms has led to the need for a tool to merge and evaluate the increasing data set sizes that result from LC-MS/MS-based shotgun proteomic experiments. MassSieve supports reports from multiple search engines with differing search characteristics, which can increase peptide sequence coverage and/or identify conflicting or ambiguous spectral assignments. PMID:20564260

The genome of the sea urchin Strongylocentrotus purpuratus.

PubMed

Sodergren, Erica; Weinstock, George M; Davidson, Eric H; Cameron, R Andrew; Gibbs, Richard A; Angerer, Robert C; Angerer, Lynne M; Arnone, Maria Ina; Burgess, David R; Burke, Robert D; Coffman, James A; Dean, Michael; Elphick, Maurice R; Ettensohn, Charles A; Foltz, Kathy R; Hamdoun, Amro; Hynes, Richard O; Klein, William H; Marzluff, William; McClay, David R; Morris, Robert L; Mushegian, Arcady; Rast, Jonathan P; Smith, L Courtney; Thorndyke, Michael C; Vacquier, Victor D; Wessel, Gary M; Wray, Greg; Zhang, Lan; Elsik, Christine G; Ermolaeva, Olga; Hlavina, Wratko; Hofmann, Gretchen; Kitts, Paul; Landrum, Melissa J; Mackey, Aaron J; Maglott, Donna; Panopoulou, Georgia; Poustka, Albert J; Pruitt, Kim; Sapojnikov, Victor; Song, Xingzhi; Souvorov, Alexandre; Solovyev, Victor; Wei, Zheng; Whittaker, Charles A; Worley, Kim; Durbin, K James; Shen, Yufeng; Fedrigo, Olivier; Garfield, David; Haygood, Ralph; Primus, Alexander; Satija, Rahul; Severson, Tonya; Gonzalez-Garay, Manuel L; Jackson, Andrew R; Milosavljevic, Aleksandar; Tong, Mark; Killian, Christopher E; Livingston, Brian T; Wilt, Fred H; Adams, Nikki; Bellé, Robert; Carbonneau, Seth; Cheung, Rocky; Cormier, Patrick; Cosson, Bertrand; Croce, Jenifer; Fernandez-Guerra, Antonio; Genevière, Anne-Marie; Goel, Manisha; Kelkar, Hemant; Morales, Julia; Mulner-Lorillon, Odile; Robertson, Anthony J; Goldstone, Jared V; Cole, Bryan; Epel, David; Gold, Bert; Hahn, Mark E; Howard-Ashby, Meredith; Scally, Mark; Stegeman, John J; Allgood, Erin L; Cool, Jonah; Judkins, Kyle M; McCafferty, Shawn S; Musante, Ashlan M; Obar, Robert A; Rawson, Amanda P; Rossetti, Blair J; Gibbons, Ian R; Hoffman, Matthew P; Leone, Andrew; Istrail, Sorin; Materna, Stefan C; Samanta, Manoj P; Stolc, Viktor; Tongprasit, Waraporn; Tu, Qiang; Bergeron, Karl-Frederik; Brandhorst, Bruce P; Whittle, James; Berney, Kevin; Bottjer, David J; Calestani, Cristina; Peterson, Kevin; Chow, Elly; Yuan, Qiu Autumn; Elhaik, Eran; Graur, Dan; Reese, Justin T; Bosdet, Ian; Heesun, Shin; Marra, Marco A; Schein, Jacqueline; Anderson, Michele K; Brockton, Virginia; Buckley, Katherine M; Cohen, Avis H; Fugmann, Sebastian D; Hibino, Taku; Loza-Coll, Mariano; Majeske, Audrey J; Messier, Cynthia; Nair, Sham V; Pancer, Zeev; Terwilliger, David P; Agca, Cavit; Arboleda, Enrique; Chen, Nansheng; Churcher, Allison M; Hallböök, F; Humphrey, Glen W; Idris, Mohammed M; Kiyama, Takae; Liang, Shuguang; Mellott, Dan; Mu, Xiuqian; Murray, Greg; Olinski, Robert P; Raible, Florian; Rowe, Matthew; Taylor, John S; Tessmar-Raible, Kristin; Wang, D; Wilson, Karen H; Yaguchi, Shunsuke; Gaasterland, Terry; Galindo, Blanca E; Gunaratne, Herath J; Juliano, Celina; Kinukawa, Masashi; Moy, Gary W; Neill, Anna T; Nomura, Mamoru; Raisch, Michael; Reade, Anna; Roux, Michelle M; Song, Jia L; Su, Yi-Hsien; Townley, Ian K; Voronina, Ekaterina; Wong, Julian L; Amore, Gabriele; Branno, Margherita; Brown, Euan R; Cavalieri, Vincenzo; Duboc, Véronique; Duloquin, Louise; Flytzanis, Constantin; Gache, Christian; Lapraz, François; Lepage, Thierry; Locascio, Annamaria; Martinez, Pedro; Matassi, Giorgio; Matranga, Valeria; Range, Ryan; Rizzo, Francesca; Röttinger, Eric; Beane, Wendy; Bradham, Cynthia; Byrum, Christine; Glenn, Tom; Hussain, Sofia; Manning, Gerard; Miranda, Esther; Thomason, Rebecca; Walton, Katherine; Wikramanayke, Athula; Wu, Shu-Yu; Xu, Ronghui; Brown, C Titus; Chen, Lili; Gray, Rachel F; Lee, Pei Yun; Nam, Jongmin; Oliveri, Paola; Smith, Joel; Muzny, Donna; Bell, Stephanie; Chacko, Joseph; Cree, Andrew; Curry, Stacey; Davis, Clay; Dinh, Huyen; Dugan-Rocha, Shannon; Fowler, Jerry; Gill, Rachel; Hamilton, Cerrissa; Hernandez, Judith; Hines, Sandra; Hume, Jennifer; Jackson, Laronda; Jolivet, Angela; Kovar, Christie; Lee, Sandra; Lewis, Lora; Miner, George; Morgan, Margaret; Nazareth, Lynne V; Okwuonu, Geoffrey; Parker, David; Pu, Ling-Ling; Thorn, Rachel; Wright, Rita

2006-11-10

We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes.

Global Monitoring of the Mammalian Lipidome by Quantitative Shotgun Lipidomics.

PubMed

Nielsen, Inger Ødum; Maeda, Kenji; Bilgin, Mesut

2017-01-01

The emerging field of lipidomics presents the systems biology approach to identify and quantify the full lipid repertoire of cells, tissues, and organisms. The importance of the lipidome is demonstrated by a number of biological studies on dysregulation of lipid metabolism in human diseases such as cancer, diabetes, and neurodegenerative diseases. Exploring changes and regulations in the huge networks of lipids and their metabolic pathways requires a lipidomics methodology: Advanced mass spectrometry that resolves the complexity of the lipidome. Here, we report a comprehensive protocol of quantitative shotgun lipidomics that enables identification and quantification of hundreds of molecular lipid species, covering a wide range of lipid classes, extracted from cultured mammalian cells.

Automated sample-preparation technologies in genome sequencing projects.

PubMed

Hilbert, H; Lauber, J; Lubenow, H; Düsterhöft, A

2000-01-01

A robotic workstation system (BioRobot 96OO, QIAGEN) and a 96-well UV spectrophotometer (Spectramax 250, Molecular Devices) were integrated in to the process of high-throughput automated sequencing of double-stranded plasmid DNA templates. An automated 96-well miniprep kit protocol (QIAprep Turbo, QIAGEN) provided high-quality plasmid DNA from shotgun clones. The DNA prepared by this procedure was used to generate more than two mega bases of final sequence data for two genomic projects (Arabidopsis thaliana and Schizosaccharomyces pombe), three thousand expressed sequence tags (ESTs) plus half a mega base of human full-length cDNA clones, and approximately 53,000 single reads for a whole genome shotgun project (Pseudomonas putida).

Comparative proteomic analysis of the aging soleus and extensor digitorum longus rat muscles using TMT labeling and mass spectrometry

PubMed Central

Chaves, Daniela F. S.; Carvalho, Paulo C.; Lima, Diogo B.; Nicastro, Humberto; Lorenzetti, Fábio M.; Filho, Mário S.; Hirabara, Sandro M.; Alves, Paulo H. M.; Moresco, James J.; Yates, John R.; Lancha, Antonio H.

2013-01-01

Sarcopenia describes an age-related decline in skeletal muscle mass, strength, and function that ultimately impairs metabolism, leads to poor balance, frequent falling, limited mobility, and a reduction in quality of life. Here we investigate the pathogenesis of sarcopenia through a proteomic shotgun approach. Briefly, we employed tandem mass tags (TMT) to quantitate and compare the protein profiles obtained from young versus old rat slow-twitch type of muscle (soleus) and a fast-twitch type of muscle (extensor digitorum longus, EDL). Our results disclose 3452 and 1848 proteins identified from soleus and EDL muscles samples of which 78 and 174 were found to be differentially expressed, respectively. In general, most of the proteins were structural related, involved in energy metabolism, oxidative stress, detoxification, or transport. Aging affected soleus and EDL muscles differently and several proteins were regulated in opposite ways. For example, pyruvate kinase had its expression and activity different in both soleus and EDL muscles. We were able to verify with existing literature many of our differentially expressed proteins as candidate aging biomarkers, and most importantly, disclose several new candidate biomarkers such as the glioblastoma amplified sequence (GAS), zero beta-globin, and prolargin. PMID:24001182

Comparative proteomic analysis of the aging soleus and extensor digitorum longus rat muscles using TMT labeling and mass spectrometry.

PubMed

Chaves, Daniela F S; Carvalho, Paulo C; Lima, Diogo B; Nicastro, Humberto; Lorenzeti, Fábio M; Siqueira-Filho, Mário; Hirabara, Sandro M; Alves, Paulo H M; Moresco, James J; Yates, John R; Lancha, Antonio H

2013-10-04

Sarcopenia describes an age-related decline in skeletal muscle mass, strength, and function that ultimately impairs metabolism and leads to poor balance, frequent falling, limited mobility, and a reduction in quality of life. Here we investigate the pathogenesis of sarcopenia through a proteomic shotgun approach. In brief, we employed tandem mass tags to quantitate and compare the protein profiles obtained from young versus old rat slow-twitch type of muscle (soleus) and a fast-twitch type of muscle (extensor digitorum longus, EDL). Our results disclose 3452 and 1848 proteins identified from soleus and EDL muscles samples, of which 78 and 174 were found to be differentially expressed, respectively. In general, most of the proteins were structural related and involved in energy metabolism, oxidative stress, detoxification, or transport. Aging affected soleus and EDL muscles differently, and several proteins were regulated in opposite ways. For example, pyruvate kinase had its expression and activity different in both soleus and EDL muscles. We were able to verify with existing literature many of our differentially expressed proteins as candidate aging biomarkers and, most importantly, disclose several new candidate biomarkers such as the glioblastoma amplified sequence, zero β-globin, and prolargin.

MIPE: A metagenome-based community structure explorer and SSU primer evaluation tool

PubMed Central

Zhou, Quan

2017-01-01

An understanding of microbial community structure is an important issue in the field of molecular ecology. The traditional molecular method involves amplification of small subunit ribosomal RNA (SSU rRNA) genes by polymerase chain reaction (PCR). However, PCR-based amplicon approaches are affected by primer bias and chimeras. With the development of high-throughput sequencing technology, unbiased SSU rRNA gene sequences can be mined from shotgun sequencing-based metagenomic or metatranscriptomic datasets to obtain a reflection of the microbial community structure in specific types of environment and to evaluate SSU primers. However, the use of short reads obtained through next-generation sequencing for primer evaluation has not been well resolved. The software MIPE (MIcrobiota metagenome Primer Explorer) was developed to adapt numerous short reads from metagenomes and metatranscriptomes. Using metagenomic or metatranscriptomic datasets as input, MIPE extracts and aligns rRNA to reveal detailed information on microbial composition and evaluate SSU rRNA primers. A mock dataset, a real Metagenomics Rapid Annotation using Subsystem Technology (MG-RAST) test dataset, two PrimerProspector test datasets and a real metatranscriptomic dataset were used to validate MIPE. The software calls Mothur (v1.33.3) and the SILVA database (v119) for the alignment and classification of rRNA genes from a metagenome or metatranscriptome. MIPE can effectively extract shotgun rRNA reads from a metagenome or metatranscriptome and is capable of classifying these sequences and exhibiting sensitivity to different SSU rRNA PCR primers. Therefore, MIPE can be used to guide primer design for specific environmental samples. PMID:28350876

Limnonectins: a new class of antimicrobial peptides from the skin secretion of the Fujian large-headed frog (Limnonectes fujianensis).

PubMed

Wu, Youjia; Wang, Lei; Zhou, Mei; Ma, Chengbang; Chen, Xiaole; Bai, Bing; Chen, Tianbao; Shaw, Chris

2011-06-01

Amphibian skin secretions are rich sources of biologically-active peptides with antimicrobial peptides predominating in many species. Several studies involving molecular cloning of biosynthetic precursor-encoding cDNAs from skin or skin secretions have revealed that these exhibit highly-conserved domain architectures with an unusually high degree of conserved nucleotide and resultant amino acid sequences within the signal peptides. This high degree of nucleotide sequence conservation has permitted the design of primers complementary to such sites facilitating "shotgun" cloning of skin or skin secretion-derived cDNA libraries from hitherto unstudied species. Here we have used such an approach using a skin secretion-derived cDNA library from an unstudied species of Chinese frog - the Fujian large-headed frog, Limnonectes fujianensis - and have discovered two 16-mer peptides of novel primary structures, named limnonectin-1Fa (SFPFFPPGICKRLKRC) and limnonectin-1Fb (SFHVFPPWMCKSLKKC), that represent the prototypes of a new class of amphibian skin antimicrobial peptide. Unusually these limnonectins display activity only against a Gram-negative bacterium (MICs of 35 and 70 μM) and are devoid of haemolytic activity at concentrations up to 160 μM. Thus the "shotgun" cloning approach described can exploit the unusually high degree of nucleotide conservation in signal peptide-encoding domains of amphibian defensive skin secretion peptide precursor-encoding cDNAs to rapidly expedite the discovery of novel and functional defensive peptides in a manner that circumvents specimen sacrifice without compromising robustness of data. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

A linear programming model for protein inference problem in shotgun proteomics.

PubMed

Huang, Ting; He, Zengyou

2012-11-15

Assembling peptides identified from tandem mass spectra into a list of proteins, referred to as protein inference, is an important issue in shotgun proteomics. The objective of protein inference is to find a subset of proteins that are truly present in the sample. Although many methods have been proposed for protein inference, several issues such as peptide degeneracy still remain unsolved. In this article, we present a linear programming model for protein inference. In this model, we use a transformation of the joint probability that each peptide/protein pair is present in the sample as the variable. Then, both the peptide probability and protein probability can be expressed as a formula in terms of the linear combination of these variables. Based on this simple fact, the protein inference problem is formulated as an optimization problem: minimize the number of proteins with non-zero probabilities under the constraint that the difference between the calculated peptide probability and the peptide probability generated from peptide identification algorithms should be less than some threshold. This model addresses the peptide degeneracy issue by forcing some joint probability variables involving degenerate peptides to be zero in a rigorous manner. The corresponding inference algorithm is named as ProteinLP. We test the performance of ProteinLP on six datasets. Experimental results show that our method is competitive with the state-of-the-art protein inference algorithms. The source code of our algorithm is available at: https://sourceforge.net/projects/prolp/. zyhe@dlut.edu.cn. Supplementary data are available at Bioinformatics Online.

Genome assembly with in vitro proximity ligation data and whole-genome triplication in lettuce

PubMed Central

Reyes-Chin-Wo, Sebastian; Wang, Zhiwen; Yang, Xinhua; Kozik, Alexander; Arikit, Siwaret; Song, Chi; Xia, Liangfeng; Froenicke, Lutz; Lavelle, Dean O.; Truco, María-José; Xia, Rui; Zhu, Shilin; Xu, Chunyan; Xu, Huaqin; Xu, Xun; Cox, Kyle; Korf, Ian; Meyers, Blake C.; Michelmore, Richard W.

2017-01-01

Lettuce (Lactuca sativa) is a major crop and a member of the large, highly successful Compositae family of flowering plants. Here we present a reference assembly for the species and family. This was generated using whole-genome shotgun Illumina reads plus in vitro proximity ligation data to create large superscaffolds; it was validated genetically and superscaffolds were oriented in genetic bins ordered along nine chromosomal pseudomolecules. We identify several genomic features that may have contributed to the success of the family, including genes encoding Cycloidea-like transcription factors, kinases, enzymes involved in rubber biosynthesis and disease resistance proteins that are expanded in the genome. We characterize 21 novel microRNAs, one of which may trigger phasiRNAs from numerous kinase transcripts. We provide evidence for a whole-genome triplication event specific but basal to the Compositae. We detect 26% of the genome in triplicated regions containing 30% of all genes that are enriched for regulatory sequences and depleted for genes involved in defence. PMID:28401891

Dryland biological soil crust cyanobacteria show unexpected decreases in abundance under long-term elevated CO2.

PubMed

Steven, Blaire; Gallegos-Graves, La Verne; Yeager, Chris M; Belnap, Jayne; Evans, R David; Kuske, Cheryl R

2012-12-01

Biological soil crusts (biocrusts) cover soil surfaces in many drylands globally. The impacts of 10 years of elevated atmospheric CO2 on the cyanobacteria in biocrusts of an arid shrubland were examined at a large manipulated experiment in Nevada, USA. Cyanobacteria-specific quantitative PCR surveys of cyanobacteria small-subunit (SSU) rRNA genes suggested a reduction in biocrust cyanobacterial biomass in the elevated CO2 treatment relative to the ambient controls. Additionally, SSU rRNA gene libraries and shotgun metagenomes showed reduced representation of cyanobacteria in the total microbial community. Taxonomic composition of the cyanobacteria was similar under ambient and elevated CO2 conditions, indicating the decline was manifest across multiple cyanobacterial lineages. Recruitment of cyanobacteria sequences from replicate shotgun metagenomes to cyanobacterial genomes representing major biocrust orders also suggested decreased abundance of cyanobacteria sequences across the majority of genomes tested. Functional assignment of cyanobacteria-related shotgun metagenome sequences indicated that four subsystem categories, three related to oxidative stress, were differentially abundant in relation to the elevated CO2 treatment. Taken together, these results suggest that elevated CO2 affected a generalized decrease in cyanobacteria in the biocrusts and may have favoured cyanobacteria with altered gene inventories for coping with oxidative stress. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Comparison of three-dimensional particle tracking and sizing using plenoptic imaging and digital in-line holography

DOE PAGES

Hall, Elise M.; Thurow, Brian S.; Guildenbecher, Daniel R.

2016-08-08

Digital in-line holography (DIH) and plenoptic photography are two techniques for single-shot, volumetric measurement of 3D particle fields. Here we present a comparison of the two methods by applying plenoptic imaging to experimental configurations that have been previously investigated with DIH. These experiments include the tracking of secondary droplets from the impact of a water drop on a thin film of water and tracking of pellets from a shotgun. Both plenoptic imaging and DIH successfully quantify the 3D nature of these particle fields. Furthermore, this includes measurement of the 3D particle position, individual particle sizes, and three-component velocity vectors. Formore » the initial processing methods presented here, both techniques give out-of-plane positional accuracy of approximately 1–2 particle diameters. For a fixed image sensor, digital holography achieves higher effective in-plane spatial resolutions. However, collimated and coherent illumination makes holography susceptible to image distortion through index of refraction gradients, as demonstrated in the shotgun experiments. In contrast, plenoptic imaging allows for a simpler experimental configuration and, due to the use of diffuse, white-light illumination, plenoptic imaging is less susceptible to image distortion in the shotgun experiments.« less

Dryland biological soil crust cyanobacteria show unexpected decreases in abundance under long-term elevated CO2

USGS Publications Warehouse

Steven, Blaire; Gallegos-Graves, La Verne; Yeager, Chris M.; Belnap, Jayne; Evans, R. David; Kuske, Cheryl R.

2012-01-01

Biological soil crusts (biocrusts) cover soil surfaces in many drylands globally. The impacts of 10 years of elevated atmospheric CO2 on the cyanobacteria in biocrusts of an arid shrubland were examined at a large manipulated experiment in Nevada, USA. Cyanobacteria-specific quantitative PCR surveys of cyanobacteria small-subunit (SSU) rRNA genes suggested a reduction in biocrust cyanobacterial biomass in the elevated CO2 treatment relative to the ambient controls. Additionally, SSU rRNA gene libraries and shotgun metagenomes showed reduced representation of cyanobacteria in the total microbial community. Taxonomic composition of the cyanobacteria was similar under ambient and elevated CO2 conditions, indicating the decline was manifest across multiple cyanobacterial lineages. Recruitment of cyanobacteria sequences from replicate shotgun metagenomes to cyanobacterial genomes representing major biocrust orders also suggested decreased abundance of cyanobacteria sequences across the majority of genomes tested. Functional assignment of cyanobacteria-related shotgun metagenome sequences indicated that four subsystem categories, three related to oxidative stress, were differentially abundant in relation to the elevated CO2 treatment. Taken together, these results suggest that elevated CO2 affected a generalized decrease in cyanobacteria in the biocrusts and may have favoured cyanobacteria with altered gene inventories for coping with oxidative stress.

How to talk about protein-level false discovery rates in shotgun proteomics.

PubMed

The, Matthew; Tasnim, Ayesha; Käll, Lukas

2016-09-01

A frequently sought output from a shotgun proteomics experiment is a list of proteins that we believe to have been present in the analyzed sample before proteolytic digestion. The standard technique to control for errors in such lists is to enforce a preset threshold for the false discovery rate (FDR). Many consider protein-level FDRs a difficult and vague concept, as the measurement entities, spectra, are manifestations of peptides and not proteins. Here, we argue that this confusion is unnecessary and provide a framework on how to think about protein-level FDRs, starting from its basic principle: the null hypothesis. Specifically, we point out that two competing null hypotheses are used concurrently in today's protein inference methods, which has gone unnoticed by many. Using simulations of a shotgun proteomics experiment, we show how confusing one null hypothesis for the other can lead to serious discrepancies in the FDR. Furthermore, we demonstrate how the same simulations can be used to verify FDR estimates of protein inference methods. In particular, we show that, for a simple protein inference method, decoy models can be used to accurately estimate protein-level FDRs for both competing null hypotheses. © 2016 The Authors. Proteomics Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Automated Finishing with Autofinish

PubMed Central

Gordon, David; Desmarais, Cindy; Green, Phil

2001-01-01

Currently, the genome sequencing community is producing shotgun sequence data at a very high rate, but finishing (collecting additional directed sequence data to close gaps and improve the quality of the data) is not matching that rate. One reason for the difference is that shotgun sequencing is highly automated but finishing is not: Most finishing decisions, such as which directed reads to obtain and which specialized sequencing techniques to use, are made by people. If finishing rates are to increase to match shotgun sequencing rates, most finishing decisions also must be automated. The Autofinish computer program (which is part of the Consed computer software package) does this by automatically choosing finishing reads. Autofinish is able to suggest most finishing reads required for completion of each sequencing project, greatly reducing the amount of human attention needed. Autofinish sometimes completely finishes the project, with no human decisions required. It cannot solve the most complex problems, so we recommend that Autofinish be allowed to suggest reads for the first three rounds of finishing, and if the project still is not finished completely, a human finisher complete the work. We compared this Autofinish-Hybrid method of finishing against a human finisher in five different projects with a variety of shotgun depths by finishing each project twice—once with each method. This comparison shows that the Autofinish-Hybrid method saves many hours over a human finisher alone, while using roughly the same number and type of reads and closing gaps at roughly the same rate. Autofinish currently is in production use at several large sequencing centers. It is designed to be adaptable to the finishing strategy of the lab—it can finish using some or all of the following: resequencing reads, reverses, custom primer walks on either subclone templates or whole clone templates, PCR, or minilibraries. Autofinish has been used for finishing cDNA, genomic clones, and whole bacterial genomes (see http://www.phrap.org). PMID:11282977

Proteome analysis of the Mycobacterium tuberculosis Beijing B0/W148 cluster

PubMed Central

Bespyatykh, Julia; Shitikov, Egor; Butenko, Ivan; Altukhov, Ilya; Alexeev, Dmitry; Mokrousov, Igor; Dogonadze, Marine; Zhuravlev, Viacheslav; Yablonsky, Peter; Ilina, Elena; Govorun, Vadim

2016-01-01

Beijing B0/W148, a “successful” clone of Mycobacterium tuberculosis, is widespread in the Russian Federation and some countries of the former Soviet Union. Here, we used label-free gel-LC-MS/MS shotgun proteomics to discover features of Beijing B0/W148 strains that could explain their success. Qualitative and quantitative proteome analyses of Beijing B0/W148 strains allowed us to identify 1,868 proteins, including 266 that were differentially abundant compared with the control strain H37Rv. To predict the biological effects of the observed differences in protein abundances, we performed Gene Ontology analysis together with analysis of protein-DNA interactions using a gene regulatory network. Our results demonstrate that Beijing B0/W148 strains have increased levels of enzymes responsible for long-chain fatty acid biosynthesis, along with a coincident decrease in the abundance of proteins responsible for their degradation. Together with high levels of HsaA (Rv3570c) protein, involved in steroid degradation, these findings provide a possible explanation for the increased transmissibility of Beijing B0/W148 strains and their survival in host macrophages. Among other, we confirmed a very low level of the SseA (Rv3283) protein in Beijing B0/W148 characteristic for all «modern» Beijing strains, which could lead to increased DNA oxidative damage, accumulation of mutations, and potentially facilitate the development of drug resistance. PMID:27356881

Phylogenetic and Functional Analysis of Metagenome Sequence from High-Temperature Archaeal Habitats Demonstrate Linkages between Metabolic Potential and Geochemistry

PubMed Central

Inskeep, William P.; Jay, Zackary J.; Herrgard, Markus J.; Kozubal, Mark A.; Rusch, Douglas B.; Tringe, Susannah G.; Macur, Richard E.; Jennings, Ryan deM.; Boyd, Eric S.; Spear, John R.; Roberto, Francisco F.

2013-01-01

Geothermal habitats in Yellowstone National Park (YNP) provide an unparalleled opportunity to understand the environmental factors that control the distribution of archaea in thermal habitats. Here we describe, analyze, and synthesize metagenomic and geochemical data collected from seven high-temperature sites that contain microbial communities dominated by archaea relative to bacteria. The specific objectives of the study were to use metagenome sequencing to determine the structure and functional capacity of thermophilic archaeal-dominated microbial communities across a pH range from 2.5 to 6.4 and to discuss specific examples where the metabolic potential correlated with measured environmental parameters and geochemical processes occurring in situ. Random shotgun metagenome sequence (∼40–45 Mb Sanger sequencing per site) was obtained from environmental DNA extracted from high-temperature sediments and/or microbial mats and subjected to numerous phylogenetic and functional analyses. Analysis of individual sequences (e.g., MEGAN and G + C content) and assemblies from each habitat type revealed the presence of dominant archaeal populations in all environments, 10 of whose genomes were largely reconstructed from the sequence data. Analysis of protein family occurrence, particularly of those involved in energy conservation, electron transport, and autotrophic metabolism, revealed significant differences in metabolic strategies across sites consistent with differences in major geochemical attributes (e.g., sulfide, oxygen, pH). These observations provide an ecological basis for understanding the distribution of indigenous archaeal lineages across high-temperature systems of YNP. PMID:23720654

Comparison of large-insert, small-insert and pyrosequencing libraries for metagenomic analysis.

PubMed

Danhorn, Thomas; Young, Curtis R; DeLong, Edward F

2012-11-01

The development of DNA sequencing methods for characterizing microbial communities has evolved rapidly over the past decades. To evaluate more traditional, as well as newer methodologies for DNA library preparation and sequencing, we compared fosmid, short-insert shotgun and 454 pyrosequencing libraries prepared from the same metagenomic DNA samples. GC content was elevated in all fosmid libraries, compared with shotgun and 454 libraries. Taxonomic composition of the different libraries suggested that this was caused by a relative underrepresentation of dominant taxonomic groups with low GC content, notably Prochlorales and the SAR11 cluster, in fosmid libraries. While these abundant taxa had a large impact on library representation, we also observed a positive correlation between taxon GC content and fosmid library representation in other low-GC taxa, suggesting a general trend. Analysis of gene category representation in different libraries indicated that the functional composition of a library was largely a reflection of its taxonomic composition, and no additional systematic biases against particular functional categories were detected at the level of sequencing depth in our samples. Another important but less predictable factor influencing the apparent taxonomic and functional library composition was the read length afforded by the different sequencing technologies. Our comparisons and analyses provide a detailed perspective on the influence of library type on the recovery of microbial taxa in metagenomic libraries and underscore the different uses and utilities of more traditional, as well as contemporary 'next-generation' DNA library construction and sequencing technologies for exploring the genomics of the natural microbial world.

Metagenomic analysis and functional characterization of the biogas microbiome using high throughput shotgun sequencing and a novel binning strategy.

PubMed

Campanaro, Stefano; Treu, Laura; Kougias, Panagiotis G; De Francisci, Davide; Valle, Giorgio; Angelidaki, Irini

2016-01-01

Biogas production is an economically attractive technology that has gained momentum worldwide over the past years. Biogas is produced by a biologically mediated process, widely known as "anaerobic digestion." This process is performed by a specialized and complex microbial community, in which different members have distinct roles in the establishment of a collective organization. Deciphering the complex microbial community engaged in this process is interesting both for unraveling the network of bacterial interactions and for applicability potential to the derived knowledge. In this study, we dissect the bioma involved in anaerobic digestion by means of high throughput Illumina sequencing (~51 gigabases of sequence data), disclosing nearly one million genes and extracting 106 microbial genomes by a novel strategy combining two binning processes. Microbial phylogeny and putative taxonomy performed using >400 proteins revealed that the biogas community is a trove of new species. A new approach based on functional properties as per network representation was developed to assign roles to the microbial species. The organization of the anaerobic digestion microbiome is resembled by a funnel concept, in which the microbial consortium presents a progressive functional specialization while reaching the final step of the process (i.e., methanogenesis). Key microbial genomes encoding enzymes involved in specific metabolic pathways, such as carbohydrates utilization, fatty acids degradation, amino acids fermentation, and syntrophic acetate oxidation, were identified. Additionally, the analysis identified a new uncultured archaeon that was putatively related to Methanomassiliicoccales but surprisingly having a methylotrophic methanogenic pathway. This study is a pioneer research on the phylogenetic and functional characterization of the microbial community populating biogas reactors. By applying for the first time high-throughput sequencing and a novel binning strategy, the identified genes were anchored to single genomes providing a clear understanding of their metabolic pathways and highlighting their involvement in anaerobic digestion. The overall research established a reference catalog of biogas microbial genomes that will greatly simplify future genomic studies.

Functional Genomics Analysis of Singapore Grouper Iridovirus: Complete Sequence Determination and Proteomic Analysis

PubMed Central

Song, Wen Jun; Qin, Qi Wei; Qiu, Jin; Huang, Can Hua; Wang, Fan; Hew, Choy Leong

2004-01-01

Here we report the complete genome sequence of Singapore grouper iridovirus (SGIV). Sequencing of the random shotgun and restriction endonuclease genomic libraries showed that the entire SGIV genome consists of 140,131 nucleotide bp. One hundred sixty-two open reading frames (ORFs) from the sense and antisense DNA strands, coding for lengths varying from 41 to 1,268 amino acids, were identified. Computer-assisted analyses of the deduced amino acid sequences revealed that 77 of the ORFs exhibited homologies to known virus genes, 23 of which matched functional iridovirus proteins. Forty-two putative conserved domains or signatures were detected in the National Center for Biotechnology Information CD-Search database and PROSITE database. An assortment of enzyme activities involved in DNA replication, transcription, nucleotide metabolism, cell signaling, etc., were identified. Viruses were cultured on a cell line derived from the embryonated egg of the grouper Epinephelus tauvina, isolated, and purified by sucrose gradient ultracentrifugation. The protein extract from the purified virions was analyzed by polyacrylamide gel electrophoresis followed by in-gel digestion of protein bands. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and database searching led to identification of 26 proteins. Twenty of these represented novel or previously unidentified genes, which were further confirmed by reverse transcription-PCR (RT-PCR) and DNA sequencing of their respective RT-PCR products. PMID:15507645

Interaction of Mycobacterium leprae with human airway epithelial cells: adherence, entry, survival, and identification of potential adhesins by surface proteome analysis.

PubMed

Silva, Carlos A M; Danelishvili, Lia; McNamara, Michael; Berredo-Pinho, Márcia; Bildfell, Robert; Biet, Franck; Rodrigues, Luciana S; Oliveira, Albanita V; Bermudez, Luiz E; Pessolani, Maria C V

2013-07-01

This study examined the in vitro interaction between Mycobacterium leprae, the causative agent of leprosy, and human alveolar and nasal epithelial cells, demonstrating that M. leprae can enter both cell types and that both are capable of sustaining bacterial survival. Moreover, delivery of M. leprae to the nasal septum of mice resulted in macrophage and epithelial cell infection in the lung tissue, sustaining the idea that the airways constitute an important M. leprae entry route into the human body. Since critical aspects in understanding the mechanisms of infection are the identification and characterization of the adhesins involved in pathogen-host cell interaction, the nude mouse-derived M. leprae cell surface-exposed proteome was studied to uncover potentially relevant adhesin candidates. A total of 279 cell surface-exposed proteins were identified based on selective biotinylation, streptavidin-affinity purification, and shotgun mass spectrometry; 11 of those proteins have been previously described as potential adhesins. In vitro assays with the recombinant forms of the histone-like protein (Hlp) and the heparin-binding hemagglutinin (HBHA), considered to be major mycobacterial adhesins, confirmed their capacity to promote bacterial attachment to epithelial cells. Taking our data together, they suggest that the airway epithelium may act as a reservoir and/or portal of entry for M. leprae in humans. Moreover, our report sheds light on the potentially critical adhesins involved in M. leprae-epithelial cell interaction that may be useful in designing more effective tools for leprosy control.

A whole-genome shotgun approach for assembling and anchoring the hexaploid bread wheat genome

DOE PAGES

Chapman, Jarrod A.; Mascher, Martin; Buluc, Aydin; ...

2015-01-31

We report that polyploid species have long been thought to be recalcitrant to whole-genome assembly. By combining high-throughput sequencing, recent developments in parallel computing, and genetic mapping, we derive, de novo, a sequence assembly representing 9.1 Gbp of the highly repetitive 16 Gbp genome of hexaploid wheat, Triticum aestivum, and assign 7.1 Gb of this assembly to chromosomal locations. The genome representation and accuracy of our assembly is comparable or even exceeds that of a chromosome-by-chromosome shotgun assembly. Our assembly and mapping strategy uses only short read sequencing technology and is applicable to any species where it is possible tomore » construct a mapping population.« less

Report on JANNAF panel on shotgun/relative quickness testing

NASA Technical Reports Server (NTRS)

Gould, R. A.

1980-01-01

As the need for more energetic solid propellants continues, a number of problems arises. One of these is the tendency of high energy propellants to transition from burning (deflagration) to detonation in regions where the propellant is present in small particle sizes; e.g., in case bonding areas of a motor after a rapid depressurization causes a shear zone at the bond interface as the stressed propellant and motor case relax at different rates. In an effort to determine the susceptibility of propellants to high strain rate break up (friability), and subsequent DDT, the propulsion community uses the shotgun/relative quickness test as one of a number of screening tests for new propellant formulations. Efforts to standardize test techniques and equipment are described.

A whole-genome shotgun approach for assembling and anchoring the hexaploid bread wheat genome

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

Chapman, Jarrod A.; Mascher, Martin; Buluc, Aydin

We report that polyploid species have long been thought to be recalcitrant to whole-genome assembly. By combining high-throughput sequencing, recent developments in parallel computing, and genetic mapping, we derive, de novo, a sequence assembly representing 9.1 Gbp of the highly repetitive 16 Gbp genome of hexaploid wheat, Triticum aestivum, and assign 7.1 Gb of this assembly to chromosomal locations. The genome representation and accuracy of our assembly is comparable or even exceeds that of a chromosome-by-chromosome shotgun assembly. Our assembly and mapping strategy uses only short read sequencing technology and is applicable to any species where it is possible tomore » construct a mapping population.« less

Bilateral shotgun pellet pulmonary emboli

PubMed Central

Huebner, Stephen; Ali, Sayed

2012-01-01

Intravascular migration of bullets and other foreign bodies is a rare but known complication of penetrating trauma. Missile embolization can represent a diagnostic challenge because it may present in various and unexpected ways. We present the case of a 54-year-old female who sustained shotgun pellet emboli to the pulmonary arteries following a left upper extremity gunshot wound and related vascular surgery. The case illustrates bilateral embolization, and the embolic events occurred following surgery. Embolization should be considered in evaluating patients with gunshot wounds, particularly if there are anomalous symptoms or the projectile is not found in the original, or expected, location. Close attention to the location of the foreign bodies on serial radiographs may reveal the diagnosis of intravascular embolization. PMID:22690290

Enrichment and proteomic analysis of plasma membrane from rat dorsal root ganglions

PubMed Central

2009-01-01

Background Dorsal root ganglion (DRG) neurons are primary sensory neurons that conduct neuronal impulses related to pain, touch and temperature senses. Plasma membrane (PM) of DRG cells plays important roles in their functions. PM proteins are main performers of the functions. However, mainly due to the very low amount of DRG that leads to the difficulties in PM sample collection, few proteomic analyses on the PM have been reported and it is a subject that demands further investigation. Results By using aqueous polymer two-phase partition in combination with high salt and high pH washing, PMs were efficiently enriched, demonstrated by western blot analysis. A total of 954 non-redundant proteins were identified from the plasma membrane-enriched preparation with CapLC-MS/MS analysis subsequent to protein separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or shotgun digestion. 205 (21.5%) of the identified proteins were unambiguously assigned as PM proteins, including a large number of signal proteins, receptors, ion channel and transporters. Conclusion The aqueous polymer two-phase partition is a simple, rapid and relatively inexpensive method. It is well suitable for the purification of PMs from small amount of tissues. Therefore, it is reasonable for the DRG PM to be enriched by using aqueous two-phase partition as a preferred method. Proteomic analysis showed that DRG PM was rich in proteins involved in the fundamental biological processes including material exchange, energy transformation and information transmission, etc. These data would help to our further understanding of the fundamental DRG functions. PMID:19889238

The Genome of the Sea Urchin Strongylocentrotus purpuratus

PubMed Central

2011-01-01

We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes. PMID:17095691

Community Structure Comparisons of Hydrothermal Vent Microbial Mats Along the Mariana Arc and Back-arc

NASA Astrophysics Data System (ADS)

Hager, K. W.; Fullerton, H.; Moyer, C. L.

2015-12-01

Hydrothermal vents along the Mariana Arc and back-arc represent a hotspot of microbial diversity that has not yet been fully recognized. The Mariana Arc and back-arc contain hydrothermal vents with varied vent effluent chemistry and temperature, which translates to diverse community composition. We have focused on iron-rich sites where the dominant primary producers are iron oxidizing bacteria. Because microbes from these environments have proven elusive in culturing efforts, we performed culture independent analysis among different microbial communities found at these hydrothermal vents. Terminal-restriction fragment length polymorphism (T-RFLP) and Illumina sequencing of small subunit ribosomal gene amplicons were used to characterize community members and identify samples for shotgun metagenomics. Used in combination, these methods will better elucidate the composition and characteristics of the bacterial communities at these hydrothermal vent systems. The overarching goal of this study is to evaluate and compare taxonomic and metabolic diversity among different communities of microbial mats. We compared communities collected on a fine scale to analyze the bacterial community based on gross mat morphology, geography, and nearby vent effluent chemistry. Taxa richness and evenness are compared with rarefaction curves to visualize diversity. As well as providing a survey of diversity this study also presents a juxtaposition of three methods in which ribosomal small subunit diversity is compared with T-RFLP, next generation amplicon sequencing, and metagenomic shotgun sequencing.

Microbial Community Profiling of Human Saliva Using Shotgun Metagenomic Sequencing

PubMed Central

Hasan, Nur A.; Young, Brian A.; Minard-Smith, Angela T.; Saeed, Kelly; Li, Huai; Heizer, Esley M.; McMillan, Nancy J.; Isom, Richard; Abdullah, Abdul Shakur; Bornman, Daniel M.; Faith, Seth A.; Choi, Seon Young; Dickens, Michael L.; Cebula, Thomas A.; Colwell, Rita R.

2014-01-01

Human saliva is clinically informative of both oral and general health. Since next generation shotgun sequencing (NGS) is now widely used to identify and quantify bacteria, we investigated the bacterial flora of saliva microbiomes of two healthy volunteers and five datasets from the Human Microbiome Project, along with a control dataset containing short NGS reads from bacterial species representative of the bacterial flora of human saliva. GENIUS, a system designed to identify and quantify bacterial species using unassembled short NGS reads was used to identify the bacterial species comprising the microbiomes of the saliva samples and datasets. Results, achieved within minutes and at greater than 90% accuracy, showed more than 175 bacterial species comprised the bacterial flora of human saliva, including bacteria known to be commensal human flora but also Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae, and Gamma proteobacteria. Basic Local Alignment Search Tool (BLASTn) analysis in parallel, reported ca. five times more species than those actually comprising the in silico sample. Both GENIUSand BLAST analyses of saliva samples identified major genera comprising the bacterial flora of saliva, but GENIUS provided a more precise description of species composition, identifying to strain in most cases and delivered results at least 10,000 times faster. Therefore, GENIUS offers a facile and accurate system for identification and quantification of bacterial species and/or strains in metagenomic samples. PMID:24846174

ProteinInferencer: Confident protein identification and multiple experiment comparison for large scale proteomics projects.

PubMed

Zhang, Yaoyang; Xu, Tao; Shan, Bing; Hart, Jonathan; Aslanian, Aaron; Han, Xuemei; Zong, Nobel; Li, Haomin; Choi, Howard; Wang, Dong; Acharya, Lipi; Du, Lisa; Vogt, Peter K; Ping, Peipei; Yates, John R

2015-11-03

Shotgun proteomics generates valuable information from large-scale and target protein characterizations, including protein expression, protein quantification, protein post-translational modifications (PTMs), protein localization, and protein-protein interactions. Typically, peptides derived from proteolytic digestion, rather than intact proteins, are analyzed by mass spectrometers because peptides are more readily separated, ionized and fragmented. The amino acid sequences of peptides can be interpreted by matching the observed tandem mass spectra to theoretical spectra derived from a protein sequence database. Identified peptides serve as surrogates for their proteins and are often used to establish what proteins were present in the original mixture and to quantify protein abundance. Two major issues exist for assigning peptides to their originating protein. The first issue is maintaining a desired false discovery rate (FDR) when comparing or combining multiple large datasets generated by shotgun analysis and the second issue is properly assigning peptides to proteins when homologous proteins are present in the database. Herein we demonstrate a new computational tool, ProteinInferencer, which can be used for protein inference with both small- or large-scale data sets to produce a well-controlled protein FDR. In addition, ProteinInferencer introduces confidence scoring for individual proteins, which makes protein identifications evaluable. This article is part of a Special Issue entitled: Computational Proteomics. Copyright © 2015. Published by Elsevier B.V.

Genomic V exons from whole genome shotgun data in reptiles.

PubMed

Olivieri, D N; von Haeften, B; Sánchez-Espinel, C; Faro, J; Gambón-Deza, F

2014-08-01

Reptiles and mammals diverged over 300 million years ago, creating two parallel evolutionary lineages amongst terrestrial vertebrates. In reptiles, two main evolutionary lines emerged: one gave rise to Squamata, while the other gave rise to Testudines, Crocodylia, and Aves. In this study, we determined the genomic variable (V) exons from whole genome shotgun sequencing (WGS) data in reptiles corresponding to the three main immunoglobulin (IG) loci and the four main T cell receptor (TR) loci. We show that Squamata lack the TRG and TRD genes, and snakes lack the IGKV genes. In representative species of Testudines and Crocodylia, the seven major IG and TR loci are maintained. As in mammals, genes of the IG loci can be grouped into well-defined IMGT clans through a multi-species phylogenetic analysis. We show that the reptilian IGHV and IGLV genes are distributed amongst the established mammalian clans, while their IGKV genes are found within a single clan, nearly exclusive from the mammalian sequences. The reptilian and mammalian TRAV genes cluster into six common evolutionary clades (since IMGT clans have not been defined for TR). In contrast, the reptilian TRBV genes cluster into three clades, which have few mammalian members. In this locus, the V exon sequences from mammals appear to have undergone different evolutionary diversification processes that occurred outside these shared reptilian clans. These sequences can be obtained in a freely available public repository (http://vgenerepertoire.org).

Application of oncoproteomics to aberrant signalling networks in changing the treatment paradigm in acute lymphoblastic leukaemia.

PubMed

López Villar, Elena; Wang, Xiangdong; Madero, Luis; Cho, William C

2015-01-01

Oncoproteomics is an important innovation in the early diagnosis, management and development of personalized treatment of acute lymphoblastic leukaemia (ALL). As inherent factors are not completely known - e.g. age or family history, radiation exposure, benzene chemical exposure, certain viral exposures such as infection with the human T-cell lymphoma/leukaemia virus-1, as well as some inherited syndromes may raise the risk of ALL - each ALL patient may modify the susceptibility of therapy. Indeed, we consider these unknown inherent factors could be explained via coupling cytogenetics plus proteomics, especially when proteins are the ones which play function within cells. Innovative proteomics to ALL therapy may help to understand the mechanism of drug resistance and toxicities, which in turn will provide some leads to improve ALL management. Most important of these are shotgun proteomic strategies to unravel ALL aberrant signalling networks. Some shotgun proteomic innovations and bioinformatic tools for ALL therapies will be discussed. As network proteins are distinctive characteristics for ALL patients, unrevealed by cytogenetics, those network proteins are currently an important source of novel therapeutic targets that emerge from shotgun proteomics. Indeed, ALL evolution can be studied for each individual patient via oncoproteomics. © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

The postman always rings twice: two cases of shotgun deaths associated with an unconventional home security alarm system.

PubMed

Asirdizer, Mahmut; Turkmen, Nursel; Akan, Okan; Yavuz, Mehmet Sunay

2014-06-01

Injury and death cases caused by booby traps are not common in forensic medicine practice. Besides, installation of booby traps including firearms is generally for suicidal and rarely for homicidal purposes. Although few patents were described about home security alarm system that were created by firearms in the United States, 1 sample of injury with a similar unconventional mechanism of home safety system was reported by Asirdizer and Yavuz in 2009. In the published case report, the story of an electrical technician who was invited to a summer house by the homeowner to check the home security alarm system was reported. In the so-called report, he was stated to be injured by the shotgun attached to the unconventional home security alarm system while checking the system. As a result, the homeowner was convicted of a possible intent to cause a life-threatening injury to the technician.The so-called homeowner and his wife died by the same shotgun attached to the same unconventional home security alarm system 4 years on from the first event. In the present case report, we have aimed to present the findings of the crime scene and the autopsies of these unusual 2 deaths and to discuss individual and legal factors in paving the way for the deaths of 2 people.

Quantitative Shotgun Proteomics Using a Uniform 15N-Labeled Standard to Monitor Proteome Dynamics in Time Course Experiments Reveals New Insights into the Heat Stress Response of Chlamydomonas reinhardtii*

PubMed Central

Mühlhaus, Timo; Weiss, Julia; Hemme, Dorothea; Sommer, Frederik; Schroda, Michael

2011-01-01

Crop-plant-yield safety is jeopardized by temperature stress caused by the global climate change. To take countermeasures by breeding and/or transgenic approaches it is essential to understand the mechanisms underlying plant acclimation to heat stress. To this end proteomics approaches are most promising, as acclimation is largely mediated by proteins. Accordingly, several proteomics studies, mainly based on two-dimensional gel-tandem MS approaches, were conducted in the past. However, results often were inconsistent, presumably attributable to artifacts inherent to the display of complex proteomes via two-dimensional-gels. We describe here a new approach to monitor proteome dynamics in time course experiments. This approach involves full 15N metabolic labeling and mass spectrometry based quantitative shotgun proteomics using a uniform 15N standard over all time points. It comprises a software framework, IOMIQS, that features batch job mediated automated peptide identification by four parallelized search engines, peptide quantification and data assembly for the processing of large numbers of samples. We have applied this approach to monitor proteome dynamics in a heat stress time course using the unicellular green alga Chlamydomonas reinhardtii as model system. We were able to identify 3433 Chlamydomonas proteins, of which 1116 were quantified in at least three of five time points of the time course. Statistical analyses revealed that levels of 38 proteins significantly increased, whereas levels of 206 proteins significantly decreased during heat stress. The increasing proteins comprise 25 (co-)chaperones and 13 proteins involved in chromatin remodeling, signal transduction, apoptosis, photosynthetic light reactions, and yet unknown functions. Proteins decreasing during heat stress were significantly enriched in functional categories that mediate carbon flux from CO2 and external acetate into protein biosynthesis, which also correlated with a rapid, but fully reversible cell cycle arrest after onset of stress. Our approach opens up new perspectives for plant systems biology and provides novel insights into plant stress acclimation. PMID:21610104

Ultra-Sensitive Elemental Analysis Using Plasmas 7.Application to Criminal Investigation

NASA Astrophysics Data System (ADS)

Suzuki, Yasuhiro

This paper describes the application of trace elemental analysis using ICP-AES and ICP-MS to criminal investigation. The comparison of trace elements, such as Rb, Sr, Zr, and so on, is effective for the forensic discrimination of glass fragments, which can be important physical evidence for connecting a suspect to a crime scene or to a victim. This procedure can be applied also to lead shotgun pellets by the removal of matrix lead as the sulfate precipitate after the dissolution of a pellet sample. The determination of a toxic element in bio-logical samples is required to prove that a victim ingested this element. Arsenous acids produced in Japan, China, Germany and Switzerland show characteristic patterns of trace elements characteristic to each country.

Metagenomics-Enabled Understanding of Soil Microbial Feedbacks to Climate Warming

NASA Astrophysics Data System (ADS)

Zhou, J.; Wu, L.; Zhili, H.; Kostas, K.; Luo, Y.; Schuur, E. A. G.; Cole, J. R.; Tiedje, J. M.

2014-12-01

Understanding the response of biological communities to climate warming is a central issue in ecology and global change biology, but it is poorly understood microbial communities. To advance system-level predictive understanding of the feedbacks of belowground microbial communities to multiple climate change factors and their impacts on soil carbon (C) and nitrogen (N) cycling processes, we have used integrated metagenomic technologies (e.g., target gene and shotgun metagenome sequencing, GeoChip, and isotope) to analyze soil microbial communities from experimental warming sites in Alaska (AK) and Oklahoma (OK), and long-term laboratory incubation. Rapid feedbacks of microbial communities to warming were observed in the AK site. Consistent with the changes in soil temperature, moisture and ecosystem respiration, microbial functional community structure was shifted after only 1.5-year warming, indicating rapid responses and high sensitivity of this permafrost ecosystem to climate warming. Also, warming stimulated not only functional genes involved in aerobic respiration of both labile and recalcitrant C, contributing to an observed 24% increase in 2010 growing season and 56% increase of decomposition of a standard substrate, but also functional genes for anaerobic processes (e.g., denitrification, sulfate reduction, methanogenesis). Further comparisons by shotgun sequencing showed significant differences of microbial community structure between AK and OK sites. The OK site was enriched in genes annotated for cellulose degradation, CO2 production, denitrification, sporulation, heat shock response, and cellular surface structures (e.g., trans-membrane transporters for glucosides), while the AK warmed plots were enriched in metabolic pathways related to labile C decomposition. Together, our results demonstrate the vulnerability of permafrost ecosystem C to climate warming and the importance of microbial feedbacks in mediating such vulnerability.

Firearms accidents in Northern India (1980-2000).

PubMed

Singh, B P

2006-01-01

This paper investigates firearms accidents from the last two decades (1980-2000), with information received from the three North Indian states, Chandigarh, Delhi and Himachal Pradesh and from the military and paramilitary organisations working in the region. In this study 139 cases were analysed for evaluation of various parameters such as type of accident, type of firearm used, age, sex, occupation and caste of victim, day of the week and the season in which the accident occurred, place of incident and location of wound. Information was extracted from the case histories and First Information Reports (FIR), and also from autopsy and injury reports. The accidents were self-inflicted in nine cases while another person fired the weapon in 130 cases. Most accidents occurred during hunting or from mishandling the firearm. Gun cleaning accidents rarely occurred and few cases were due to technical defects of the firearm. Shotguns and rifles caused most of the accidents. Shotgun accidents occurred mostly in rural areas whereas the cases involving rifles and handguns were from urban areas. Victims were predominantly male with a mean age of 26 and 30 years for males and females respectively. Victims' occupations were principally agricultural followed by service class and housewives. The most aggressive community was the Kshatriya caste. Most of the accidents occurred during the winter season and on days between Sunday and Tuesday. Accident locations were most commonly the forest, the fields or the home. The chest and head were the most common anatomical sites of injury. All accidental cases were the result of only one firing. It is important to mention that, based on these findings, firearm accidents could be reduced significantly if firearm holders were better trained regarding the handling and safety aspects of their guns, particularly farmers to whom firearm licences are granted specifically for the protection of crops.

Production of haemolysins by strains of the Actinobacillus minor/"porcitonsillarum" complex.

PubMed

Arya, Gitanjali; Niven, Donald F

2010-03-24

Actinobacillus minor and "Actinobacillus porcitonsillarum" are distinguished by their haemolytic activities, the latter organism being haemolytic and the former, non-haemolytic. Analysis of a whole genome shotgun sequence, however, revealed that A. minor strain 202, like "A. porcitonsillarum", possesses a haemolysin-encoding apxII operon. The purpose of this study was therefore to investigate haemolysin production by this organism and also by three additional members of the A. minor/"porcitonsillarum" complex, strains 33PN and 7ATS and A. minor strain NM305(T). Primers based on sequences within the apxII genes of strain 202 allowed the amplification of appropriately sized fragments from DNA from strain 33PN suggesting that this organism also possesses an apxII operon. Analysis of a whole genome shotgun sequence failed to reveal any trace of an apxII operon in strain NM305(T) and attempts to amplify apxII genes from DNA from strain 7ATS also failed. Strains 202 and 33PN, and surprisingly, the type strain of A. minor and strain 7ATS, were all found to be haemolysin-positive as growth media from cultures of these organisms could promote the lysis of erythrocytes in suspension. The erythrocyte specificities of the haemolysins produced by strains 202 and 33PN indicated that the haemolytic activities exhibited by these organisms were due to ApxII. In keeping with the apparent lack of apxII genes in strains NM305(T) and 7ATS, the haemolysins produced by these organisms were not erythrocyte-specific and with both organisms, haemolytic activity appeared to be due to a combination of heat-stable and heat-labile components. The identities of these components, however, remain unknown. Copyright 2009 Elsevier B.V. All rights reserved.

Microbiological profile of chicken carcasses: A comparative analysis using shotgun metagenomic sequencing

PubMed Central

Cesare, Alessandra De; Palma, Federica; Lucchi, Alex; Pasquali, Frederique; Manfreda, Gerardo

2018-01-01

In the last few years metagenomic and 16S rRNA sequencing have completly changed the microbiological investigations of food products. In this preliminary study, the microbiological profile of chicken carcasses collected from animals fed with different diets were tested by using shotgun metagenomic sequencing. A total of 15 carcasses have been collected at the slaughetrhouse at the end of the refrigeration tunnel from chickens reared for 35 days and fed with a control diet (n=5), a diet supplemented with 1500 FTU/kg of commercial phytase (n=5) and a diet supplemented with 1500 FTU/kg of commercial phytase and 3g/kg of inositol (n=5). Ten grams of neck and breast skin were obtained from each carcass and submited to total DNA extraction by using the DNeasy Blood & Tissue Kit (Qiagen). Sequencing libraries have been prepared by using the Nextera XT DNA Library Preparation Kit (Illumina) and sequenced in a HiScanSQ (Illumina) at 100 bp in paired ends. A number of sequences ranging between 5 and 9 million was obtained for each sample. Sequence analysis showed that Proteobacteria and Firmicutes represented more than 98% of whole bacterial populations associated to carcass skin in all groups but their abundances were different between groups. Moraxellaceae and other degradative bacteria showed a significantly higher abundance in the control compared to the treated groups. Furthermore, Clostridium perfringens showed a relative frequency of abundance significantly higher in the group fed with phytase and Salmonella enterica in the group fed with phytase plus inositol. The results of this preliminary study showed that metagenome sequencing is suitable to investigate and monitor carcass microbiota in order to detect specific pathogenic and/or degradative populations. PMID:29732327

Analysis of Illumina Microbial Assemblies

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

Clum, Alicia; Foster, Brian; Froula, Jeff

2010-05-28

Since the emerging of second generation sequencing technologies, the evaluation of different sequencing approaches and their assembly strategies for different types of genomes has become an important undertaken. Next generation sequencing technologies dramatically increase sequence throughput while decreasing cost, making them an attractive tool for whole genome shotgun sequencing. To compare different approaches for de-novo whole genome assembly, appropriate tools and a solid understanding of both quantity and quality of the underlying sequence data are crucial. Here, we performed an in-depth analysis of short-read Illumina sequence assembly strategies for bacterial and archaeal genomes. Different types of Illumina libraries as wellmore » as different trim parameters and assemblers were evaluated. Results of the comparative analysis and sequencing platforms will be presented. The goal of this analysis is to develop a cost-effective approach for the increased throughput of the generation of high quality microbial genomes.« less

32 CFR 552.119 - Registration and storage.

Code of Federal Regulations, 2012 CFR

2012-07-01

... include rifles, shotguns, handguns and antique firearms owned by personnel residing on Fort Lewis Military... Weapons Registration Section when clearing post. Upon any sale or transfer of a registered weapon, the...

32 CFR 552.119 - Registration and storage.

Code of Federal Regulations, 2010 CFR

2010-07-01

... include rifles, shotguns, handguns and antique firearms owned by personnel residing on Fort Lewis Military... Weapons Registration Section when clearing post. Upon any sale or transfer of a registered weapon, the...

32 CFR 552.119 - Registration and storage.

Code of Federal Regulations, 2011 CFR

2011-07-01

... include rifles, shotguns, handguns and antique firearms owned by personnel residing on Fort Lewis Military... Weapons Registration Section when clearing post. Upon any sale or transfer of a registered weapon, the...

Effects of 28 days of resistance exercise while consuming commercially available pre- and post-workout supplements, NO-Shotgun® and NO-Synthesize® on body composition, muscle strength and mass, markers of protein synthesis, and clinical safety markers in males

PubMed Central

2011-01-01

Purpose The effects of 28 days of heavy resistance training while ingesting the pre- and post-workout supplements, NO-Shotgun® and NO-Synthesize® were determined on body composition, muscle strength and mass, markers of protein synthesis, and clinical safety markers. Methods Nineteen non-resistance-trained males participated in a resistance training program 4 times/week for 28 days while either ingesting 27 g/day of carbohydrate (CARB) or NO-Shotgun® 30 min pre-exercise and 27 g/day of carbohydrate or NO- Synthesize® 30 min post-exercise (NOSS). Data were analyzed with separate 2 × 2 ANOVA (p < 0.05). Results Total body mass was increased in both groups (p = 0.001), but not different between groups. Fat mass was unchanged with CARB, but NOSS decreased fat mass (p = 0.026). Both groups increased fat-free mass (p = 0.001); however, the increases were greater with NOSS (p = 0.023). NOSS underwent greater increases in upper-body (p = 0.023) and lower-body (p = 0.035) strength than CARB. Myofibrillar protein significantly increased in both groups (p = 0.041), with NOSS being greater than CARB (p = 0.049). All of the MHC isoforms were significantly increased in both groups; however, NOSS was greater than CARB for MHC 1 (p = 0.013) and MHC 2A (p = 0.046). All of the myogenic regulatory factors were significantly increased in both groups; however, NOSS was greater than CARB for Myo-D (p = 0.038) and MRF-4 (p = 0.001). For the whole blood and serum clinical chemistry markers, all variables remained within normal clinical ranges. Conclusions Heavy resistance training for 28 days, with NO-Shotgun® and NO-Synthesize® ingested before and after exercise, respectively, significantly improved body composition and increased muscle mass and performance without abnormally impacting any of the clinical chemistry markers. PMID:22050827

A review of lead poisoning in swans

USGS Publications Warehouse

Blus, L.J.

1994-01-01

Nearly 10,000 swans of six species or subspecies from 14 countries have died from poisoning caused by lead that originated from ingestion of fishing weights, shotgun pellets (shot), or contaminated vegetation or sediments associated with mining and smelting wastes. Lead contamination in mute swans in England caused local population declines during the late 1970s and 1980s. More tundra swans died from lead poisoning than any other species. The extreme record involved an estimated 7200 tundra swans that died over five winters at one locality in North Carolina. The recent legislation to ban lead fishing weights in most of England and Wales and recent replacement of lead shot with steel shot for waterfowl hunting in the United States and a few areas of Europe, including Denmark, are expected to reduce the incidence of lead poisoning in swans.

Improved Understanding of Microbial Iron and Sulfate Reduction Through a Combination of Bottom-up and Top-down Functional Proteomics Assays

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

Richardson, Ruth

Our overall goal was to improve the understanding of microbial iron and sulfate reduction by evaluating a diverse iron and sulfate reducing organisms utilizing a multi-omics approach combining “top-down” and “bottom-up” omics methodologies. We initiated one of the first combined comparative genomics, shotgun proteomics, RTqPCR, and heterologous expression studies in pursuit of our project objectives. Within the first year of this project, we created a new bioinformatics tool for ortholog identification (“SPOCS”). SPOCS is described in our publication, Curtis et al., 2013. Using this tool we were able to identify conserved orthologous groups across diverse iron and sulfate reducing microorganismsmore » from Firmicutes, gamma-proteobacteria and delta-proteobacteria. For six iron and sulfate reducers we also performed shotgun proteomics (“bottom-up” proteomics including accurate mass and time (AMT) tag and iTRAQ approaches). Cultures include Gram (-) and Gram (+) microbes. Gram (-) were: Geobacter sulfureducens (grown on iron citrate and fumarate), Geobacter bemidjiensis (grown on iron citrate and fumarate), Shewanella oneidiensis (grown on iron citrate and fumarate) and Anaeromyxobacter dehalogenans (grown on iron citrate and fumarate). Although all cultures grew on insoluble iron, the iron precipitates interfered with protein extraction and analysis; which remains a major challenge for researchers in disparate study systems. Among the Gram (-) organisms studied, Anaeromyxobacter dehalogenans remains the most poorly characterized. Yet, it is arguably the most versatile organisms we studied. In this work we have used comparative proteomics to hypothesize which two of the dozens of predicted c-type cytochromes within Anaeromyxobacter dehalogenans may be directly involved in soluble iron reduction. Unfortunately, heterologous expression of these Anaeromyxobacter dehalogenans ctype cytochromes led to poor protein production and/or formation of inclusion bodies, even when we co-expressed several genes known to be important for assembly of cytochrome holoenzymes. We confirmed the proteomics trends at the RNA level by designing specific primer sets for hypothesized iron reductases in Anaeromyxobacter dehalogenans, and performed Reverse Transcription-qPCR. AD_0127 was 20 fold upregulated only on iron citrate conditions. AD_0127 is described as a hypothetical protein, but Pfam predicts it to be C554 type cytochrome having a possible role in nitrification (Wang et al., in preparation).« less

Comparative Analysis of the Peanut Witches'-Broom Phytoplasma Genome Reveals Horizontal Transfer of Potential Mobile Units and Effectors

PubMed Central

Lo, Wen-Sui; Lin, Chan-Pin; Kuo, Chih-Horng

2013-01-01

Phytoplasmas are a group of bacteria that are associated with hundreds of plant diseases. Due to their economical importance and the difficulties involved in the experimental study of these obligate pathogens, genome sequencing and comparative analysis have been utilized as powerful tools to understand phytoplasma biology. To date four complete phytoplasma genome sequences have been published. However, these four strains represent limited phylogenetic diversity. In this study, we report the shotgun sequencing and evolutionary analysis of a peanut witches'-broom (PnWB) phytoplasma genome. The availability of this genome provides the first representative of the 16SrII group and substantially improves the taxon sampling to investigate genome evolution. The draft genome assembly contains 13 chromosomal contigs with a total size of 562,473 bp, covering ∼90% of the chromosome. Additionally, a complete plasmid sequence is included. Comparisons among the five available phytoplasma genomes reveal the differentiations in gene content and metabolic capacity. Notably, phylogenetic inferences of the potential mobile units (PMUs) in these genomes indicate that horizontal transfer may have occurred between divergent phytoplasma lineages. Because many effectors are associated with PMUs, the horizontal transfer of these transposon-like elements can contribute to the adaptation and diversification of these pathogens. In summary, the findings from this study highlight the importance of improving taxon sampling when investigating genome evolution. Moreover, the currently available sequences are inadequate to fully characterize the pan-genome of phytoplasmas. Future genome sequencing efforts to expand phylogenetic diversity are essential in improving our understanding of phytoplasma evolution. PMID:23626855

Proteomic analysis of a NAP1 Clostridium difficile clinical isolate resistant to metronidazole.

PubMed

Chong, Patrick M; Lynch, Tarah; McCorrister, Stuart; Kibsey, Pamela; Miller, Mark; Gravel, Denise; Westmacott, Garrett R; Mulvey, Michael R

2014-01-01

Clostridium difficile is an anaerobic, Gram-positive bacterium that has been implicated as the leading cause of antibiotic-associated diarrhea. Metronidazole is currently the first-line treatment for mild to moderate C. difficile infections. Our laboratory isolated a strain of C. difficile with a stable resistance phenotype to metronidazole. A shotgun proteomics approach was used to compare differences in the proteomes of metronidazole-resistant and -susceptible isolates. NAP1 C. difficile strains CD26A54_R (Met-resistant), CD26A54_S (reduced- susceptibility), and VLOO13 (Met-susceptible) were grown to mid-log phase, and spiked with metronidazole at concentrations 2 doubling dilutions below the MIC. Peptides from each sample were labeled with iTRAQ and subjected to 2D-LC-MS/MS analysis. In the absence of metronidazole, higher expression was observed of some proteins in C. difficile strains CD26A54_S and CD26A54_R that may be involved with reduced susceptibility or resistance to metronidazole, including DNA repair proteins, putative nitroreductases, and the ferric uptake regulator (Fur). After treatment with metronidazole, moderate increases were seen in the expression of stress-related proteins in all strains. A moderate increase was also observed in the expression of the DNA repair protein RecA in CD26A54_R. This study provided an in-depth proteomic analysis of a stable, metronidazole-resistant C. difficile isolate. The results suggested that a multi-factorial response may be associated with high level metronidazole-resistance in C. difficile, including the possible roles of altered iron metabolism and/or DNA repair.

Forest harvesting reduces the soil metagenomic potential for biomass decomposition.

PubMed

Cardenas, Erick; Kranabetter, J M; Hope, Graeme; Maas, Kendra R; Hallam, Steven; Mohn, William W

2015-11-01

Soil is the key resource that must be managed to ensure sustainable forest productivity. Soil microbial communities mediate numerous essential ecosystem functions, and recent studies show that forest harvesting alters soil community composition. From a long-term soil productivity study site in a temperate coniferous forest in British Columbia, 21 forest soil shotgun metagenomes were generated, totaling 187 Gb. A method to analyze unassembled metagenome reads from the complex community was optimized and validated. The subsequent metagenome analysis revealed that, 12 years after forest harvesting, there were 16% and 8% reductions in relative abundances of biomass decomposition genes in the organic and mineral soil layers, respectively. Organic and mineral soil layers differed markedly in genetic potential for biomass degradation, with the organic layer having greater potential and being more strongly affected by harvesting. Gene families were disproportionately affected, and we identified 41 gene families consistently affected by harvesting, including families involved in lignin, cellulose, hemicellulose and pectin degradation. The results strongly suggest that harvesting profoundly altered below-ground cycling of carbon and other nutrients at this site, with potentially important consequences for forest regeneration. Thus, it is important to determine whether these changes foreshadow long-term changes in forest productivity or resilience and whether these changes are broadly characteristic of harvested forests.

Metagenomics analysis of microbial communities associated with a traditional rice wine starter culture (Xaj-pitha) of Assam, India.

PubMed

Bora, Sudipta Sankar; Keot, Jyotshna; Das, Saurav; Sarma, Kishore; Barooah, Madhumita

2016-12-01

This is the first report on the microbial diversity of xaj-pitha, a rice wine fermentation starter culture through a metagenomics approach involving Illumine-based whole genome shotgun (WGS) sequencing method. Metagenomic DNA was extracted from rice wine starter culture concocted by Ahom community of Assam and analyzed using a MiSeq ® System. A total of 2,78,231 contigs, with an average read length of 640.13 bp, were obtained. Data obtained from the use of several taxonomic profiling tools were compared with previously reported microbial diversity studies through the culture-dependent and culture-independent method. The microbial community revealed the existence of amylase producers, such as Rhizopus delemar, Mucor circinelloides, and Aspergillus sp. Ethanol producers viz., Meyerozyma guilliermondii, Wickerhamomyces ciferrii, Saccharomyces cerevisiae, Candida glabrata, Debaryomyces hansenii, Ogataea parapolymorpha, and Dekkera bruxellensis, were found associated with the starter culture along with a diverse range of opportunistic contaminants. The bacterial microflora was dominated by lactic acid bacteria (LAB). The most frequent occurring LAB was Lactobacillus plantarum, Lactobacillus brevis, Leuconostoc lactis, Weissella cibaria, Lactococcus lactis, Weissella para mesenteroides, Leuconostoc pseudomesenteroides, etc. Our study provided a comprehensive picture of microbial diversity associated with rice wine fermentation starter and indicated the superiority of metagenomic sequencing over previously used techniques.

Characterization of the reniform nematode genome by shotgun sequencing.

PubMed

Nyaku, Seloame T; Sripathi, Venkateswara R; Kantety, Ramesh V; Cseke, Sarah B; Buyyarapu, Ramesh; Mc Ewan, Robert; Gu, Yong Q; Lawrence, Kathy; Senwo, Zachary; Sripathi, Padmini; George, Pheba; Sharma, Govind C

2014-04-01

The reniform nematode (RN), a major agricultural pest particularly on cotton in the United States, is among the major plant-parasitic nematodes for which limited genomic information exists. In this study, over 380 Mb of sequence data were generated from pooled DNA of four adult female RNs and assembled into 67,317 contigs, including 25,904 (38.5%) predicted coding contigs and 41,413 (61.5%) noncoding contigs. Most of the characterized repeats were of low complexity (88.9%), and 0.9% of the contigs matched with 53.2% of GenBank ESTs. The most frequent Gene Ontology (GO) terms for molecular function and biological process were protein binding (32%) and embryonic development (20%). Further analysis showed that 741 (1.1%), 94 (0.1%), and 169 (0.25%) RN genomic contigs matched with 1328 (13.9%), 1480 (5.4%), and 1330 (7.4%) supercontigs of Meloidogyne incognita, Brugia malayi, and Pristionchus pacificus, respectively. Chromosome 5 of Caenorhabditis elegans had the highest number of hits to the RN contigs. Seven putative detoxification genes and three carbohydrate-active enzymes (CAZymes) involved in cell wall degradation were studied in more detail. Additionally, kinases, G protein-coupled receptors, and neuropeptides functioning in physiological, developmental, and regulatory processes were identified in the RN genome.

Evaluation of microbial community in hydrothermal field by direct DNA sequencing

NASA Astrophysics Data System (ADS)

Kawarabayasi, Y.; Maruyama, A.

2002-12-01

Many extremophiles have been discovered from terrestrial and marine hydrothermal fields. Some thermophiles can grow beyond 90°C in culture, while direct microscopic analysis occasionally indicates that microbes may survive in much hotter hydrothermal fluids. However, it is very difficult to isolate and cultivate such microbes from the environments, i.e., over 99% of total microbes remains undiscovered. Based on experiences of entire microbial genome analysis (Y.K.) and microbial community analysis (A.M.), we started to find out unique microbes/genes in hydrothermal fields through direct sequencing of environmental DNA fragments. At first, shotgun plasmid libraries were directly constructed with the DNA molecules prepared from mixed microbes collected by an in situ filtration system from low-temperature fluids at RM24 in the Southern East Pacific Rise (S-EPR). A gene amplification (PCR) technique was not used for preventing mutation in the process. The nucleotide sequences of 285 clones indicated that no sequence had identical data in public databases. Among 27 clones determined entire sequences, no ORF was identified on 14 clones like intron in Eukaryote. On four clones, tetra-nucleotide-long multiple tandem repetitive sequences were identified. This type of sequence was identified in some familiar disease in human. The result indicates that living/dead materials with eukaryotic features may exist in this low temperature field. Secondly, shotgun plasmid libraries were constructed from the environmental DNA prepared from Beppu hot springs. In randomly-selected 143 clones used for sequencing, no known sequence was identified. Unlike the clones in S-EPR library, clear ORFs were identified on all nine clones determined the entire sequence. It was found that one clone, H4052, contained the complete Aspartyl-tRNA synthetase. Phylogenetic analysis using amino acid sequences of this gene indicated that this gene was separated from other Euryarchaea before the differentiation of species. Thus, some novel archaeal species are expected to be in this field. The present direct cloning and sequencing technique is now opening a window to the new world in hydrothermal microbial community analysis.

The Combined Use of Proteomics and Transcriptomics Reveals a Complex Secondary Metabolite Network in Peperomia obtusifolia.

PubMed

Batista, Andrea N L; Santos-Pinto, José Roberto A Dos; Batista, João M; Souza-Moreira, Tatiana M; Santoni, Mariana M; Zanelli, Cleslei F; Kato, Massuo J; López, Silvia N; Palma, Mario S; Furlan, Maysa

2017-05-26

Peperomia obtusifolia, an ornamental plant from the Piperaceae family, accumulates a series of secondary metabolites with interesting biological properties. From a biosynthesis standpoint, this species produces several benzopyrans derived from orsellinic acid, which is a polyketide typically found in fungi. Additionally, the chiral benzopyrans were reported as racemic and/or as diastereomeric mixtures, which raises questions about the level of enzymatic control in the cyclization step for the formation of the 3,4-dihydro-2H-pyran moiety. Therefore, this article describes the use of shotgun proteomic and transcriptome studies as well as phytochemical profiling for the characterization of the main biosynthesis pathways active in P. obtusifolia. This combined approach resulted in the identification of a series of proteins involved in its secondary metabolism, including tocopherol cyclase and prenyltransferases. The activity of these enzymes was supported by the phytochemical profiling performed in different organs of P. obtusifolia. However, the polyketide synthases possibly involved in the production of orsellinic acid could not be identified, suggesting that orsellinic acid may be produced by endophytes intimately associated with the plant.

50 CFR 92.20 - Methods and means.

Code of Federal Regulations, 2010 CFR

2010-10-01

... birds: (a) Swivel guns, shotguns larger than 10 gauge, punt guns, battery guns, machine guns, fish hooks... traditional means of harvesting auklets on Diomede and St. Lawrence islands.); (e) Hunting with the aid of...

32 CFR 552.119 - Registration and storage.

Code of Federal Regulations, 2013 CFR

2013-07-01

... personal weapons to include rifles, shotguns, handguns and antique firearms owned by personnel residing on...) will be turned in at the Weapons Registration Section when clearing post. Upon any sale or transfer of...

32 CFR 552.119 - Registration and storage.

Code of Federal Regulations, 2014 CFR

2014-07-01

... personal weapons to include rifles, shotguns, handguns and antique firearms owned by personnel residing on...) will be turned in at the Weapons Registration Section when clearing post. Upon any sale or transfer of...

Development of 13 microsatellites for Gunnison Sage-grouse (Centrocercus minimus) using next-generation shotgun sequencing and their utility in Greater Sage-grouse (Centrocercus urophasianus)

USGS Publications Warehouse

Fike, Jennifer A.; Oyler-McCance, Sara J.; Zimmerman, Shawna J; Castoe, Todd A.

2015-01-01

Gunnison Sage-grouse are an obligate sagebrush species that has experienced significant population declines and has been proposed for listing under the U.S. Endangered Species Act. In order to examine levels of connectivity among Gunnison Sage-grouse leks, we identified 13 novel microsatellite loci though next-generation shotgun sequencing, and tested them on the closely related Greater Sage-grouse. The number of alleles per locus ranged from 2 to 12. No loci were found to be linked, although 2 loci revealed significant departures from Hardy–Weinberg equilibrium or evidence of null alleles. While these microsatellites were designed for Gunnison Sage-grouse, they also work well for Greater Sage-grouse and could be used for numerous genetic questions including landscape and population genetics.

Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood

PubMed Central

Fan, H. Christina; Blumenfeld, Yair J.; Chitkara, Usha; Hudgins, Louanne; Quake, Stephen R.

2008-01-01

We directly sequenced cell-free DNA with high-throughput shotgun sequencing technology from plasma of pregnant women, obtaining, on average, 5 million sequence tags per patient sample. This enabled us to measure the over- and underrepresentation of chromosomes from an aneuploid fetus. The sequencing approach is polymorphism-independent and therefore universally applicable for the noninvasive detection of fetal aneuploidy. Using this method, we successfully identified all nine cases of trisomy 21 (Down syndrome), two cases of trisomy 18 (Edward syndrome), and one case of trisomy 13 (Patau syndrome) in a cohort of 18 normal and aneuploid pregnancies; trisomy was detected at gestational ages as early as the 14th week. Direct sequencing also allowed us to study the characteristics of cell-free plasma DNA, and we found evidence that this DNA is enriched for sequences from nucleosomes. PMID:18838674

[The role of the comprehensive approach for the characteristic of the destructive effect of a non-lethal cartridge with two rubber bullets].

PubMed

Stragis, V B; Makarov, I Yu; Karelin, V V; Shevchuk, D Yu; Chechenin, E S

This article was designed to report the results of forensic medical, criminalistics, and comprehensive expertise of the subject who suffered from a non-perforating shotgun wound affecting the soft tissues and blood vessels in the femoral region. It was shown that only the scrutinous comprehensive full-scale expert examination of the injured site in the framework of forensic medical expertise makes it possible to exclude the probability of the expert error and formulate the reliable and substantiated conclusion as regards the fact and the conditions of the shotgun injury by a concrete type of the cartridge (e.g. having a caliber equal to 410/76 Stopper-2) with two spherical rubber bullets fired from a known weapon (Saiga-410S hunting carbine).

Deterministic protein inference for shotgun proteomics data provides new insights into Arabidopsis pollen development and function

PubMed Central

Grobei, Monica A.; Qeli, Ermir; Brunner, Erich; Rehrauer, Hubert; Zhang, Runxuan; Roschitzki, Bernd; Basler, Konrad; Ahrens, Christian H.; Grossniklaus, Ueli

2009-01-01

Pollen, the male gametophyte of flowering plants, represents an ideal biological system to study developmental processes, such as cell polarity, tip growth, and morphogenesis. Upon hydration, the metabolically quiescent pollen rapidly switches to an active state, exhibiting extremely fast growth. This rapid switch requires relevant proteins to be stored in the mature pollen, where they have to retain functionality in a desiccated environment. Using a shotgun proteomics approach, we unambiguously identified ∼3500 proteins in Arabidopsis pollen, including 537 proteins that were not identified in genetic or transcriptomic studies. To generate this comprehensive reference data set, which extends the previously reported pollen proteome by a factor of 13, we developed a novel deterministic peptide classification scheme for protein inference. This generally applicable approach considers the gene model–protein sequence–protein accession relationships. It allowed us to classify and eliminate ambiguities inherently associated with any shotgun proteomics data set, to report a conservative list of protein identifications, and to seamlessly integrate data from previous transcriptomics studies. Manual validation of proteins unambiguously identified by a single, information-rich peptide enabled us to significantly reduce the false discovery rate, while keeping valuable identifications of shorter and lower abundant proteins. Bioinformatic analyses revealed a higher stability of pollen proteins compared to those of other tissues and implied a protein family of previously unknown function in vesicle trafficking. Interestingly, the pollen proteome is most similar to that of seeds, indicating physiological similarities between these developmentally distinct tissues. PMID:19546170

Nontarget bird exposure to DRC-1339 during fall in North Dakota and spring in South Dakota

USGS Publications Warehouse

Custer, Thomas W.; Custer, Christine M.; Dummer, Paul M.; Linz, George M.; Sileo, Louis; Stahl, Randal S.; Johnston, John J.; Linz, G.M.

2003-01-01

Blackbirds frequently use ripening sunflower (Heltantbus annuus) as a food source in the northern Great Plains. In 1999 and 2000, the avicide DRC-1339 (3-chloro-4-methylaniline hydrochloride) was used experimentally on fall-ripening sunflower fields in North Dakota so researchers could evaluate its effectiveness for reducing crop depredations by blackbirds. DRC-1339 was applied to rice and broadcast on the ground in a confined area within ripening sunflower fields. One objective of this study was to determine whether nontarget birds, birds other than blackbirds, were eating rice and were exposed to the DRC-1339. In 1999, 8 of 11 (73%) sparrows collected by shotgun in sunflower fields treated with DRe-1339 had rice in their gastrointestinal (GI) tracts. In 2000, 5 mourning doves (Zenaida macroura) and 3 sparrows were collected by shotgun in sunflower fields treated with DRC-1339. Three doves had rice in their GI tracts, 4 doves and all 3 sparrows had measurable DRC1339 concentrations in their GI tracts, and 3 mourning doves and 1 savannah sparrow (Passerculus sanduncbensis) exhibited histopathological signs of kidney damage. In April 2002, untreated rice was applied to corn stubble plots in South Dakota to determine which bird species ate rice. In 2002, 3 of 3 song sparrows (Melospiza melodia) collected by shotgun had rice in their GI tracts. Our results demonstrate that the use of DRC-1339 to control blackbirds in the northern Great Plains will likely expose nontarget birds to the DRC-1339 bait.

A Preliminary Comparison of Three Dimensional Particle Tracking and Sizing using Plenoptic Imaging and Digital In-line Holography

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

Guildenbecher, Daniel Robert; Munz, Elise Dahnke; Farias, Paul Abraham

2015-12-01

Digital in-line holography and plenoptic photography are two techniques for single-shot, volumetric measurement of 3D particle fields. Here we present a preliminary comparison of the two methods by applying plenoptic imaging to experimental configurations that have been previously investigated with digital in-line holography. These experiments include the tracking of secondary droplets from the impact of a water drop on a thin film of water and tracking of pellets from a shotgun. Both plenoptic imaging and digital in-line holography successfully quantify the 3D nature of these particle fields. This includes measurement of the 3D particle position, individual particle sizes, and three-componentmore » velocity vectors. For the initial processing methods presented here, both techniques give out-of-plane positional accuracy of approximately 1-2 particle diameters. For a fixed image sensor, digital holography achieves higher effective in-plane spatial resolutions. However, collimated and coherent illumination makes holography susceptible to image distortion through index of refraction gradients, as demonstrated in the shotgun experiments. On the other hand, plenotpic imaging allows for a simpler experimental configuration. Furthermore, due to the use of diffuse, white-light illumination, plenoptic imaging is less susceptible to image distortion in the shotgun experiments. Additional work is needed to better quantify sources of uncertainty, particularly in the plenoptic experiments, as well as develop data processing methodologies optimized for the plenoptic measurement.« less

A Preliminary Comparison of Three Dimensional Particle Tracking and Sizing using Plenoptic Imaging and Digital In-line Holography [PowerPoint

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

Guildenbecher, Daniel Robert; Munz, Elise Dahnke; Farias, Paul Abraham

2015-12-01

Digital in-line holography and plenoptic photography are two techniques for single-shot, volumetric measurement of 3D particle fields. Here we present a preliminary comparison of the two methods by applying plenoptic imaging to experimental configurations that have been previously investigated with digital in-line holography. These experiments include the tracking of secondary droplets from the impact of a water drop on a thin film of water and tracking of pellets from a shotgun. Both plenoptic imaging and digital in-line holography successfully quantify the 3D nature of these particle fields. This includes measurement of the 3D particle position, individual particle sizes, and three-componentmore » velocity vectors. For the initial processing methods presented here, both techniques give out-of-plane positional accuracy of approximately 1-2 particle diameters. For a fixed image sensor, digital holography achieves higher effective in-plane spatial resolutions. However, collimated and coherent illumination makes holography susceptible to image distortion through index of refraction gradients, as demonstrated in the shotgun experiments. On the other hand, plenotpic imaging allows for a simpler experimental configuration. Furthermore, due to the use of diffuse, white-light illumination, plenoptic imaging is less susceptible to image distortion in the shotgun experiments. Additional work is needed to better quantify sources of uncertainty, particularly in the plenoptic experiments, as well as develop data processing methodologies optimized for the plenoptic measurement.« less

Integrated shotgun sequencing and bioinformatics pipeline allows ultra-fast mitogenome recovery and confirms substantial gene rearrangements in Australian freshwater crayfishes

PubMed Central

2014-01-01

Background Although it is possible to recover the complete mitogenome directly from shotgun sequencing data, currently reported methods and pipelines are still relatively time consuming and costly. Using a sample of the Australian freshwater crayfish Engaeus lengana, we demonstrate that it is possible to achieve three-day turnaround time (four hours hands-on time) from tissue sample to NCBI-ready submission file through the integration of MiSeq sequencing platform, Nextera sample preparation protocol, MITObim assembly algorithm and MITOS annotation pipeline. Results The complete mitochondrial genome of the parastacid freshwater crayfish, Engaeus lengana, was recovered by modest shotgun sequencing (1.2 giga bases) using the Illumina MiSeq benchtop sequencing platform. Genome assembly using the MITObim mitogenome assembler recovered the mitochondrial genome as a single contig with a 97-fold mean coverage (min. = 17; max. = 138). The mitogenome consists of 15,934 base pairs and contains the typical 37 mitochondrial genes and a non-coding AT-rich region. The genome arrangement is similar to the only other published parastacid mitogenome from the Australian genus Cherax. Conclusions We infer that the gene order arrangement found in Cherax destructor is common to Australian crayfish and may be a derived feature of the southern hemisphere family Parastacidae. Further, we report to our knowledge, the simplest and fastest protocol for the recovery and assembly of complete mitochondrial genomes using the MiSeq benchtop sequencer. PMID:24484414

Diversity of thermophiles in a Malaysian hot spring determined using 16S rRNA and shotgun metagenome sequencing.

PubMed

Chan, Chia Sing; Chan, Kok-Gan; Tay, Yea-Ling; Chua, Yi-Heng; Goh, Kian Mau

2015-01-01

The Sungai Klah (SK) hot spring is the second hottest geothermal spring in Malaysia. This hot spring is a shallow, 150-m-long, fast-flowing stream, with temperatures varying from 50 to 110°C and a pH range of 7.0-9.0. Hidden within a wooded area, the SK hot spring is continually fed by plant litter, resulting in a relatively high degree of total organic content (TOC). In this study, a sample taken from the middle of the stream was analyzed at the 16S rRNA V3-V4 region by amplicon metagenome sequencing. Over 35 phyla were detected by analyzing the 16S rRNA data. Firmicutes and Proteobacteria represented approximately 57% of the microbiome. Approximately 70% of the detected thermophiles were strict anaerobes; however, Hydrogenobacter spp., obligate chemolithotrophic thermophiles, represented one of the major taxa. Several thermophilic photosynthetic microorganisms and acidothermophiles were also detected. Most of the phyla identified by 16S rRNA were also found using the shotgun metagenome approaches. The carbon, sulfur, and nitrogen metabolism within the SK hot spring community were evaluated by shotgun metagenome sequencing, and the data revealed diversity in terms of metabolic activity and dynamics. This hot spring has a rich diversified phylogenetic community partly due to its natural environment (plant litter, high TOC, and a shallow stream) and geochemical parameters (broad temperature and pH range). It is speculated that symbiotic relationships occur between the members of the community.

In-Source Fragmentation and the Sources of Partially Tryptic Peptides in Shotgun Proteomics

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

Kim, Jong-Seo; Monroe, Matthew E.; Camp, David G.

2013-02-01

Partially tryptic peptides are often identified in shotgun proteomics using trypsin as the proteolytic enzyme; however, it has been controversial regarding the sources of such partially tryptic peptides. Herein we investigate the impact of in-source fragmentation on shotgun proteomics using three biological samples, including a standard protein mixture, a mouse brain tissue homogenate, and a mouse plasma sample. Since the in-source fragments of a peptide retain the same elution time with its parent fully tryptic peptide, the partially tryptic peptides from in-source fragmentation can be distinguished from the other partially tryptic peptides by plotting the observed retention times against themore » computationally predicted retention times. Most partially tryptic in-source fragmentation artifacts were misaligned from the linear distribution of fully tryptic peptides. The impact of in-source fragmentation on peptide identifications was clearly significant in a less complex sample such as a standard protein digest, where ~60 % of unique peptides were observed as partially tryptic peptides from in-source fragmentation. In mouse brain or mouse plasma samples, in-source fragmentation contributed to 1-3 % of all identified peptides. The other major source of partially tryptic peptides in complex biological samples is presumably proteolytic processing by endogenous proteases in the samples. By filtering out the in-source fragmentation artifacts from the identified partially tryptic or non-tryptic peptides, it is possible to directly survey in-vivo proteolytic processing in biological samples such as blood plasma.« less

Complementary Proteome and Transcriptome Profiling in Developing Grains of a Notched-Belly Rice Mutant Reveals Key Pathways Involved in Chalkiness Formation

PubMed Central

Lin, Zhaomiao; Wang, Zunxin; Zhang, Xincheng; Li, Ganghua; Wang, Shaohua; Ding, Yanfeng

2017-01-01

Rice grain chalkiness is a highly complex trait involved in multiple metabolic pathways and controlled by polygenes and growth conditions. To uncover novel aspects of chalkiness formation, we performed an integrated profiling of gene activity in the developing grains of a notched-belly rice mutant. Using exhaustive tandem mass spectrometry-based shotgun proteomics and whole-genome RNA sequencing to generate a nearly complete catalog of expressed mRNAs and proteins, we reliably identified 38,476 transcripts and 3,840 proteins. Comparison between the translucent part and chalky part of the notched-belly grains resulted in only a few differently express genes (240) and differently express proteins (363), thus making it possible to focus on ‘core’ genes or common pathways. Several novel key pathways were identified as of relevance to chalkiness formation, in particular the shift of C and N metabolism, the down-regulation of ribosomal proteins and the resulting low abundance of storage proteins especially the 13 kDa prolamin subunit, and the suppressed photosynthetic capacity in the pericarp of the chalky part. Further, genes and proteins as transporters for carbohydrates, amino acid/peptides, proteins, lipids and inorganic ions showed an increasing expression pattern in the chalky part of the notched-belly grains. Similarly, transcripts and proteins of receptors for auxin, ABA, ethylene and brassinosteroid were also up-regulated. In summary, this joint analysis of transcript and protein profiles provides a comprehensive reference map of gene activity regarding the physiological state in the chalky endosperm. PMID:28158863

Interaction of Mycobacterium leprae with Human Airway Epithelial Cells: Adherence, Entry, Survival, and Identification of Potential Adhesins by Surface Proteome Analysis

PubMed Central

Silva, Carlos A. M.; Danelishvili, Lia; McNamara, Michael; Berredo-Pinho, Márcia; Bildfell, Robert; Biet, Franck; Rodrigues, Luciana S.; Oliveira, Albanita V.

2013-01-01

This study examined the in vitro interaction between Mycobacterium leprae, the causative agent of leprosy, and human alveolar and nasal epithelial cells, demonstrating that M. leprae can enter both cell types and that both are capable of sustaining bacterial survival. Moreover, delivery of M. leprae to the nasal septum of mice resulted in macrophage and epithelial cell infection in the lung tissue, sustaining the idea that the airways constitute an important M. leprae entry route into the human body. Since critical aspects in understanding the mechanisms of infection are the identification and characterization of the adhesins involved in pathogen-host cell interaction, the nude mouse-derived M. leprae cell surface-exposed proteome was studied to uncover potentially relevant adhesin candidates. A total of 279 cell surface-exposed proteins were identified based on selective biotinylation, streptavidin-affinity purification, and shotgun mass spectrometry; 11 of those proteins have been previously described as potential adhesins. In vitro assays with the recombinant forms of the histone-like protein (Hlp) and the heparin-binding hemagglutinin (HBHA), considered to be major mycobacterial adhesins, confirmed their capacity to promote bacterial attachment to epithelial cells. Taking our data together, they suggest that the airway epithelium may act as a reservoir and/or portal of entry for M. leprae in humans. Moreover, our report sheds light on the potentially critical adhesins involved in M. leprae-epithelial cell interaction that may be useful in designing more effective tools for leprosy control. PMID:23670556

Deregulation of focal adhesion formation and cytoskeletal tension due to loss of A-type lamins.

PubMed

Corne, Tobias D J; Sieprath, Tom; Vandenbussche, Jonathan; Mohammed, Danahe; Te Lindert, Mariska; Gevaert, Kris; Gabriele, Sylvain; Wolf, Katarina; De Vos, Winnok H

2017-09-03

The nuclear lamina mechanically integrates the nucleus with the cytoskeleton and extracellular environment and regulates gene expression. These functions are exerted through direct and indirect interactions with the lamina's major constituent proteins, the A-type lamins, which are encoded by the LMNA gene. Using quantitative stable isotope labeling-based shotgun proteomics we have analyzed the proteome of human dermal fibroblasts in which we have depleted A-type lamins by means of a sustained siRNA-mediated LMNA knockdown. Gene ontology analysis revealed that the largest fraction of differentially produced proteins was involved in actin cytoskeleton organization, in particular proteins involved in focal adhesion dynamics, such as actin-related protein 2 and 3 (ACTR2/3), subunits of the ARP2/3 complex, and fascin actin-bundling protein 1 (FSCN1). Functional validation using quantitative immunofluorescence showed a significant reduction in the size of focal adhesion points in A-type lamin depleted cells, which correlated with a reduction in early cell adhesion capacity and an increased cell motility. At the same time, loss of A-type lamins led to more pronounced stress fibers and higher traction forces. This phenotype could not be mimicked or reversed by experimental modulation of the STAT3-IL6 pathway, but it was partly recapitulated by chemical inhibition of the ARP2/3 complex. Thus, our data suggest that the loss of A-type lamins perturbs the balance between focal adhesions and cytoskeletal tension. This imbalance may contribute to mechanosensing defects observed in certain laminopathies.

Genomic, proteomic, and biochemical analysis of the organohalide respiratory pathway in Desulfitobacterium dehalogenans.

PubMed

Kruse, Thomas; van de Pas, Bram A; Atteia, Ariane; Krab, Klaas; Hagen, Wilfred R; Goodwin, Lynne; Chain, Patrick; Boeren, Sjef; Maphosa, Farai; Schraa, Gosse; de Vos, Willem M; van der Oost, John; Smidt, Hauke; Stams, Alfons J M

2015-03-01

Desulfitobacterium dehalogenans is able to grow by organohalide respiration using 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) as an electron acceptor. We used a combination of genome sequencing, biochemical analysis of redox active components, and shotgun proteomics to study elements of the organohalide respiratory electron transport chain. The genome of Desulfitobacterium dehalogenans JW/IU-DC1(T) consists of a single circular chromosome of 4,321,753 bp with a GC content of 44.97%. The genome contains 4,252 genes, including six rRNA operons and six predicted reductive dehalogenases. One of the reductive dehalogenases, CprA, is encoded by a well-characterized cprTKZEBACD gene cluster. Redox active components were identified in concentrated suspensions of cells grown on formate and Cl-OHPA or formate and fumarate, using electron paramagnetic resonance (EPR), visible spectroscopy, and high-performance liquid chromatography (HPLC) analysis of membrane extracts. In cell suspensions, these components were reduced upon addition of formate and oxidized after addition of Cl-OHPA, indicating involvement in organohalide respiration. Genome analysis revealed genes that likely encode the identified components of the electron transport chain from formate to fumarate or Cl-OHPA. Data presented here suggest that the first part of the electron transport chain from formate to fumarate or Cl-OHPA is shared. Electrons are channeled from an outward-facing formate dehydrogenase via menaquinones to a fumarate reductase located at the cytoplasmic face of the membrane. When Cl-OHPA is the terminal electron acceptor, electrons are transferred from menaquinones to outward-facing CprA, via an as-yet-unidentified membrane complex, and potentially an extracellular flavoprotein acting as an electron shuttle between the quinol dehydrogenase membrane complex and CprA. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Genomic, Proteomic, and Biochemical Analysis of the Organohalide Respiratory Pathway in Desulfitobacterium dehalogenans

PubMed Central

van de Pas, Bram A.; Atteia, Ariane; Krab, Klaas; Hagen, Wilfred R.; Goodwin, Lynne; Chain, Patrick; Boeren, Sjef; Maphosa, Farai; Schraa, Gosse; de Vos, Willem M.; van der Oost, John; Smidt, Hauke

2014-01-01

Desulfitobacterium dehalogenans is able to grow by organohalide respiration using 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) as an electron acceptor. We used a combination of genome sequencing, biochemical analysis of redox active components, and shotgun proteomics to study elements of the organohalide respiratory electron transport chain. The genome of Desulfitobacterium dehalogenans JW/IU-DC1T consists of a single circular chromosome of 4,321,753 bp with a GC content of 44.97%. The genome contains 4,252 genes, including six rRNA operons and six predicted reductive dehalogenases. One of the reductive dehalogenases, CprA, is encoded by a well-characterized cprTKZEBACD gene cluster. Redox active components were identified in concentrated suspensions of cells grown on formate and Cl-OHPA or formate and fumarate, using electron paramagnetic resonance (EPR), visible spectroscopy, and high-performance liquid chromatography (HPLC) analysis of membrane extracts. In cell suspensions, these components were reduced upon addition of formate and oxidized after addition of Cl-OHPA, indicating involvement in organohalide respiration. Genome analysis revealed genes that likely encode the identified components of the electron transport chain from formate to fumarate or Cl-OHPA. Data presented here suggest that the first part of the electron transport chain from formate to fumarate or Cl-OHPA is shared. Electrons are channeled from an outward-facing formate dehydrogenase via menaquinones to a fumarate reductase located at the cytoplasmic face of the membrane. When Cl-OHPA is the terminal electron acceptor, electrons are transferred from menaquinones to outward-facing CprA, via an as-yet-unidentified membrane complex, and potentially an extracellular flavoprotein acting as an electron shuttle between the quinol dehydrogenase membrane complex and CprA. PMID:25512312

Hair combing to collect organic gunshot residues (OGSR).

PubMed

MacCrehan, William A; Layman, Malinda J; Secl, Janelle D

2003-08-12

A protocol is presented for the collection and analysis of gunshot residues (GSR) from hair. A fine-toothed comb is used for collection of the residues. A small zip-closure bag serves as a container for both sample storage and extraction of the characteristic organic powder additives. The success of this residue recovery approach was tested on simulated shooters and victims using mannequin-supported human wig hair as well as on human shooters. Residues were collected from four weapons: a revolver and semi-automatic pistol, rifle and shotgun. One characteristic additive, nitroglycerin, was detected by capillary electrophoresis (CE) in the majority of the collection experiments.

A Combination of Histological, Physiological, and Proteomic Approaches Shed Light on Seed Desiccation Tolerance of the Basal Angiosperm Amborella trichopoda.

PubMed

Villegente, Matthieu; Marmey, Philippe; Job, Claudette; Galland, Marc; Cueff, Gwendal; Godin, Béatrice; Rajjou, Loïc; Balliau, Thierry; Zivy, Michel; Fogliani, Bruno; Sarramegna-Burtet, Valérie; Job, Dominique

2017-07-28

Desiccation tolerance allows plant seeds to remain viable in a dry state for years and even centuries. To reveal potential evolutionary processes of this trait, we have conducted a shotgun proteomic analysis of isolated embryo and endosperm from mature seeds of Amborella trichopoda , an understory shrub endemic to New Caledonia that is considered to be the basal extant angiosperm. The present analysis led to the characterization of 415 and 69 proteins from the isolated embryo and endosperm tissues, respectively. The role of these proteins is discussed in terms of protein evolution and physiological properties of the rudimentary, underdeveloped, Amborella embryos, notably considering that the acquisition of desiccation tolerance corresponds to the final developmental stage of mature seeds possessing large embryos.

A Combination of Histological, Physiological, and Proteomic Approaches Shed Light on Seed Desiccation Tolerance of the Basal Angiosperm Amborella trichopoda

PubMed Central

Villegente, Matthieu; Marmey, Philippe; Job, Claudette; Galland, Marc; Cueff, Gwendal; Godin, Béatrice; Rajjou, Loïc; Balliau, Thierry; Zivy, Michel; Sarramegna-Burtet, Valérie; Job, Dominique

2017-01-01

Desiccation tolerance allows plant seeds to remain viable in a dry state for years and even centuries. To reveal potential evolutionary processes of this trait, we have conducted a shotgun proteomic analysis of isolated embryo and endosperm from mature seeds of Amborella trichopoda, an understory shrub endemic to New Caledonia that is considered to be the basal extant angiosperm. The present analysis led to the characterization of 415 and 69 proteins from the isolated embryo and endosperm tissues, respectively. The role of these proteins is discussed in terms of protein evolution and physiological properties of the rudimentary, underdeveloped, Amborella embryos, notably considering that the acquisition of desiccation tolerance corresponds to the final developmental stage of mature seeds possessing large embryos. PMID:28788068

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

Denef, Vincent; Shah, Manesh B; Verberkmoes, Nathan C

The recent surge in microbial genomic sequencing, combined with the development of high-throughput liquid chromatography-mass-spectrometry-based (LC/LC-MS/MS) proteomics, has raised the question of the extent to which genomic information of one strain or environmental sample can be used to profile proteomes of related strains or samples. Even with decreasing sequencing costs, it remains impractical to obtain genomic sequence for every strain or sample analyzed. Here, we evaluate how shotgun proteomics is affected by amino acid divergence between the sample and the genomic database using a probability-based model and a random mutation simulation model constrained by experimental data. To assess the effectsmore » of nonrandom distribution of mutations, we also evaluated identification levels using in silico peptide data from sequenced isolates with average amino acid identities (AAI) varying between 76 and 98%. We compared the predictions to experimental protein identification levels for a sample that was evaluated using a database that included genomic information for the dominant organism and for a closely related variant (95% AAI). The range of models set the boundaries at which half of the proteins in a proteomic experiment can be identified to be 77-92% AAI between orthologs in the sample and database. Consistent with this prediction, experimental data indicated loss of half the identifiable proteins at 90% AAI. Additional analysis indicated a 6.4% reduction of the initial protein coverage per 1% amino acid divergence and total identification loss at 86% AAI. Consequently, shotgun proteomics is capable of cross-strain identifications but avoids most crossspecies false positives.« less

Directed Shotgun Proteomics Guided by Saturated RNA-seq Identifies a Complete Expressed Prokaryotic Proteome

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

Omasits, U.; Quebatte, Maxime; Stekhoven, Daniel J.

2013-11-01

Prokaryotes, due to their moderate complexity, are particularly amenable to the comprehensive identification of the protein repertoire expressed under different conditions. We applied a generic strategy to identify a complete expressed prokaryotic proteome, which is based on the analysis of RNA and proteins extracted from matched samples. Saturated transcriptome profiling by RNA-seq provided an endpoint estimate of the protein-coding genes expressed under two conditions which mimic the interaction of Bartonella henselae with its mammalian host. Directed shotgun proteomics experiments were carried out on four subcellular fractions. By specifically targeting proteins which are short, basic, low abundant, and membrane localized, wemore » could eliminate their initial underrepresentation compared to the estimated endpoint. A total of 1250 proteins were identified with an estimated false discovery rate below 1%. This represents 85% of all distinct annotated proteins and ~90% of the expressed protein-coding genes. Genes that were detected at the transcript but not protein level, were found to be highly enriched in several genomic islands. Furthermore, genes that lacked an ortholog and a functional annotation were not detected at the protein level; these may represent examples of overprediction in genome annotations. A dramatic membrane proteome reorganization was observed, including differential regulation of autotransporters, adhesins, and hemin binding proteins. Particularly noteworthy was the complete membrane proteome coverage, which included expression of all members of the VirB/D4 type IV secretion system, a key virulence factor.« less

Functional Glycomic Analysis of Human Milk Glycans Reveals the Presence of Virus Receptors and Embryonic Stem Cell Biomarkers*

PubMed Central

Yu, Ying; Mishra, Shreya; Song, Xuezheng; Lasanajak, Yi; Bradley, Konrad C.; Tappert, Mary M.; Air, Gillian M.; Steinhauer, David A.; Halder, Sujata; Cotmore, Susan; Tattersall, Peter; Agbandje-McKenna, Mavis; Cummings, Richard D.; Smith, David F.

2012-01-01

Human milk contains a large diversity of free glycans beyond lactose, but their functions are not well understood. To explore their functional recognition, here we describe a shotgun glycan microarray prepared from isolated human milk glycans (HMGs), and our studies on their recognition by viruses, antibodies, and glycan-binding proteins (GBPs), including lectins. The total neutral and sialylated HMGs were derivatized with a bifunctional fluorescent tag, separated by multidimensional HPLC, and archived in a tagged glycan library, which was then used to print a shotgun glycan microarray (SGM). This SGM was first interrogated with well defined GBPs and antibodies. These data demonstrated both the utility of the array and provided preliminary structural information (metadata) about this complex glycome. Anti-TRA-1 antibodies that recognize human pluripotent stem cells specifically recognized several HMGs that were then further structurally defined as novel epitopes for these antibodies. Human influenza viruses and Parvovirus Minute Viruses of Mice also specifically recognized several HMGs. For glycan sequencing, we used a novel approach termed metadata-assisted glycan sequencing (MAGS), in which we combine information from analyses of glycans by mass spectrometry with glycan interactions with defined GBPs and antibodies before and after exoglycosidase treatments on the microarray. Together, these results provide novel insights into diverse recognition functions of HMGs and show the utility of the SGM approach and MAGS as resources for defining novel glycan recognition by GBPs, antibodies, and pathogens. PMID:23115247

Directed shotgun proteomics guided by saturated RNA-seq identifies a complete expressed prokaryotic proteome

PubMed Central

Omasits, Ulrich; Quebatte, Maxime; Stekhoven, Daniel J.; Fortes, Claudia; Roschitzki, Bernd; Robinson, Mark D.; Dehio, Christoph; Ahrens, Christian H.

2013-01-01

Prokaryotes, due to their moderate complexity, are particularly amenable to the comprehensive identification of the protein repertoire expressed under different conditions. We applied a generic strategy to identify a complete expressed prokaryotic proteome, which is based on the analysis of RNA and proteins extracted from matched samples. Saturated transcriptome profiling by RNA-seq provided an endpoint estimate of the protein-coding genes expressed under two conditions which mimic the interaction of Bartonella henselae with its mammalian host. Directed shotgun proteomics experiments were carried out on four subcellular fractions. By specifically targeting proteins which are short, basic, low abundant, and membrane localized, we could eliminate their initial underrepresentation compared to the estimated endpoint. A total of 1250 proteins were identified with an estimated false discovery rate below 1%. This represents 85% of all distinct annotated proteins and ∼90% of the expressed protein-coding genes. Genes that were detected at the transcript but not protein level, were found to be highly enriched in several genomic islands. Furthermore, genes that lacked an ortholog and a functional annotation were not detected at the protein level; these may represent examples of overprediction in genome annotations. A dramatic membrane proteome reorganization was observed, including differential regulation of autotransporters, adhesins, and hemin binding proteins. Particularly noteworthy was the complete membrane proteome coverage, which included expression of all members of the VirB/D4 type IV secretion system, a key virulence factor. PMID:23878158

Structural Analysis of Unsaturated Glycosphingolipids Using Shotgun Ozone-Induced Dissociation Mass Spectrometry

NASA Astrophysics Data System (ADS)

Barrientos, Rodell C.; Vu, Ngoc; Zhang, Qibin

2017-08-01

Glycosphingolipids are essential biomolecules widely distributed across biological kingdoms yet remain relatively underexplored owing to both compositional and structural complexity. While the glycan head group has been the subject of most studies, there is paucity of reports on the lipid moiety, particularly the location of unsaturation. In this paper, ozone-induced dissociation mass spectrometry (OzID-MS) implemented in a traveling wave-based quadrupole time-of-flight (Q-ToF) mass spectrometer was applied to study unsaturated glycosphingolipids using shotgun approach. Resulting high resolution mass spectra facilitated the unambiguous identification of diagnostic OzID product ions. Using [M+Na]+ adducts of authentic standards, we observed that the long chain base and fatty acyl unsaturation had distinct reactivity with ozone. The reactivity of unsaturation in the fatty acyl chain was about 8-fold higher than that in the long chain base, which enables their straightforward differentiation. Influence of the head group, fatty acyl hydroxylation, and length of fatty acyl chain on the oxidative cleavage of double bonds was also observed. Application of this technique to bovine brain galactocerebrosides revealed co-isolated isobaric and regioisomeric species, which otherwise would be incompletely identified using contemporary collision-induced dissociation (CID) alone. These results highlight the potential of OzID-MS in glycosphingolipids research, which not only provides complementary structural information to existing CID technique but also facilitates de novo structural determination of these complex biomolecules. [Figure not available: see fulltext.

Statistical Analysis of the Processes Controlling Choline and Ethanolamine Glycerophospholipid Molecular Species Composition

PubMed Central

Kiebish, Michael A.; Yang, Kui; Han, Xianlin; Gross, Richard W.; Chuang, Jeffrey

2012-01-01

The regulation and maintenance of the cellular lipidome through biosynthetic, remodeling, and catabolic mechanisms are critical for biological homeostasis during development, health and disease. These complex mechanisms control the architectures of lipid molecular species, which have diverse yet highly regulated fatty acid chains at both the sn1 and sn2 positions. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) serve as the predominant biophysical scaffolds in membranes, acting as reservoirs for potent lipid signals and regulating numerous enzymatic processes. Here we report the first rigorous computational dissection of the mechanisms influencing PC and PE molecular architectures from high-throughput shotgun lipidomic data. Using novel statistical approaches, we have analyzed multidimensional mass spectrometry-based shotgun lipidomic data from developmental mouse heart and mature mouse heart, lung, brain, and liver tissues. We show that in PC and PE, sn1 and sn2 positions are largely independent, though for low abundance species regulatory processes may interact with both the sn1 and sn2 chain simultaneously, leading to cooperative effects. Chains with similar biochemical properties appear to be remodeled similarly. We also see that sn2 positions are more regulated than sn1, and that PC exhibits stronger cooperative effects than PE. A key aspect of our work is a novel statistically rigorous approach to determine cooperativity based on a modified Fisher's exact test using Markov Chain Monte Carlo sampling. This computational approach provides a novel tool for developing mechanistic insight into lipidomic regulation. PMID:22662143

LTR Retrotransposons Contribute to Genomic Gigantism in Plethodontid Salamanders

PubMed Central

Sun, Cheng; Shepard, Donald B.; Chong, Rebecca A.; López Arriaza, José; Hall, Kathryn; Castoe, Todd A.; Feschotte, Cédric; Pollock, David D.; Mueller, Rachel Lockridge

2012-01-01

Among vertebrates, most of the largest genomes are found within the salamanders, a clade of amphibians that includes 613 species. Salamander genome sizes range from ∼14 to ∼120 Gb. Because genome size is correlated with nucleus and cell sizes, as well as other traits, morphological evolution in salamanders has been profoundly affected by genomic gigantism. However, the molecular mechanisms driving genomic expansion in this clade remain largely unknown. Here, we present the first comparative analysis of transposable element (TE) content in salamanders. Using high-throughput sequencing, we generated genomic shotgun data for six species from the Plethodontidae, the largest family of salamanders. We then developed a pipeline to mine TE sequences from shotgun data in taxa with limited genomic resources, such as salamanders. Our summaries of overall TE abundance and diversity for each species demonstrate that TEs make up a substantial portion of salamander genomes, and that all of the major known types of TEs are represented in salamanders. The most abundant TE superfamilies found in the genomes of our six focal species are similar, despite substantial variation in genome size. However, our results demonstrate a major difference between salamanders and other vertebrates: salamander genomes contain much larger amounts of long terminal repeat (LTR) retrotransposons, primarily Ty3/gypsy elements. Thus, the extreme increase in genome size that occurred in salamanders was likely accompanied by a shift in TE landscape. These results suggest that increased proliferation of LTR retrotransposons was a major molecular mechanism contributing to genomic expansion in salamanders. PMID:22200636

Introducing the CPL/MUW proteome database: interpretation of human liver and liver cancer proteome profiles by referring to isolated primary cells.

PubMed

Wimmer, Helge; Gundacker, Nina C; Griss, Johannes; Haudek, Verena J; Stättner, Stefan; Mohr, Thomas; Zwickl, Hannes; Paulitschke, Verena; Baron, David M; Trittner, Wolfgang; Kubicek, Markus; Bayer, Editha; Slany, Astrid; Gerner, Christopher

2009-06-01

Interpretation of proteome data with a focus on biomarker discovery largely relies on comparative proteome analyses. Here, we introduce a database-assisted interpretation strategy based on proteome profiles of primary cells. Both 2-D-PAGE and shotgun proteomics are applied. We obtain high data concordance with these two different techniques. When applying mass analysis of tryptic spot digests from 2-D gels of cytoplasmic fractions, we typically identify several hundred proteins. Using the same protein fractions, we usually identify more than thousand proteins by shotgun proteomics. The data consistency obtained when comparing these independent data sets exceeds 99% of the proteins identified in the 2-D gels. Many characteristic differences in protein expression of different cells can thus be independently confirmed. Our self-designed SQL database (CPL/MUW - database of the Clinical Proteomics Laboratories at the Medical University of Vienna accessible via www.meduniwien.ac.at/proteomics/database) facilitates (i) quality management of protein identification data, which are based on MS, (ii) the detection of cell type-specific proteins and (iii) of molecular signatures of specific functional cell states. Here, we demonstrate, how the interpretation of proteome profiles obtained from human liver tissue and hepatocellular carcinoma tissue is assisted by the Clinical Proteomics Laboratories at the Medical University of Vienna-database. Therefore, we suggest that the use of reference experiments supported by a tailored database may substantially facilitate data interpretation of proteome profiling experiments.

ReprDB and panDB: minimalist databases with maximal microbial representation.

PubMed

Zhou, Wei; Gay, Nicole; Oh, Julia

2018-01-18

Profiling of shotgun metagenomic samples is hindered by a lack of unified microbial reference genome databases that (i) assemble genomic information from all open access microbial genomes, (ii) have relatively small sizes, and (iii) are compatible to various metagenomic read mapping tools. Moreover, computational tools to rapidly compile and update such databases to accommodate the rapid increase in new reference genomes do not exist. As a result, database-guided analyses often fail to profile a substantial fraction of metagenomic shotgun sequencing reads from complex microbiomes. We report pipelines that efficiently traverse all open access microbial genomes and assemble non-redundant genomic information. The pipelines result in two species-resolution microbial reference databases of relatively small sizes: reprDB, which assembles microbial representative or reference genomes, and panDB, for which we developed a novel iterative alignment algorithm to identify and assemble non-redundant genomic regions in multiple sequenced strains. With the databases, we managed to assign taxonomic labels and genome positions to the majority of metagenomic reads from human skin and gut microbiomes, demonstrating a significant improvement over a previous database-guided analysis on the same datasets. reprDB and panDB leverage the rapid increases in the number of open access microbial genomes to more fully profile metagenomic samples. Additionally, the databases exclude redundant sequence information to avoid inflated storage or memory space and indexing or analyzing time. Finally, the novel iterative alignment algorithm significantly increases efficiency in pan-genome identification and can be useful in comparative genomic analyses.

27 CFR 479.192 - Commerce in firearms and ammunition.

Code of Federal Regulations, 2010 CFR

2010-04-01

..., FIREARMS, AND EXPLOSIVES, DEPARTMENT OF JUSTICE FIREARMS AND AMMUNITION MACHINE GUNS, DESTRUCTIVE DEVICES..., machine guns, short-barreled shotguns, or short-barreled rifles, see 18 U.S.C. Chapter 44, and Part 478 of...

27 CFR 479.192 - Commerce in firearms and ammunition.

Code of Federal Regulations, 2012 CFR

2012-04-01

..., FIREARMS, AND EXPLOSIVES, DEPARTMENT OF JUSTICE FIREARMS AND AMMUNITION MACHINE GUNS, DESTRUCTIVE DEVICES..., machine guns, short-barreled shotguns, or short-barreled rifles, see 18 U.S.C. Chapter 44, and Part 478 of...

27 CFR 479.192 - Commerce in firearms and ammunition.

Code of Federal Regulations, 2014 CFR

2014-04-01

..., FIREARMS, AND EXPLOSIVES, DEPARTMENT OF JUSTICE FIREARMS AND AMMUNITION MACHINE GUNS, DESTRUCTIVE DEVICES..., machine guns, short-barreled shotguns, or short-barreled rifles, see 18 U.S.C. Chapter 44, and Part 478 of...

27 CFR 479.192 - Commerce in firearms and ammunition.

Code of Federal Regulations, 2013 CFR

2013-04-01

..., FIREARMS, AND EXPLOSIVES, DEPARTMENT OF JUSTICE FIREARMS AND AMMUNITION MACHINE GUNS, DESTRUCTIVE DEVICES..., machine guns, short-barreled shotguns, or short-barreled rifles, see 18 U.S.C. Chapter 44, and Part 478 of...

27 CFR 479.192 - Commerce in firearms and ammunition.

Code of Federal Regulations, 2011 CFR

2011-04-01

..., FIREARMS, AND EXPLOSIVES, DEPARTMENT OF JUSTICE FIREARMS AND AMMUNITION MACHINE GUNS, DESTRUCTIVE DEVICES..., machine guns, short-barreled shotguns, or short-barreled rifles, see 18 U.S.C. Chapter 44, and Part 478 of...

EBI metagenomics in 2016 - an expanding and evolving resource for the analysis and archiving of metagenomic data

PubMed Central

Mitchell, Alex; Bucchini, Francois; Cochrane, Guy; Denise, Hubert; Hoopen, Petra ten; Fraser, Matthew; Pesseat, Sebastien; Potter, Simon; Scheremetjew, Maxim; Sterk, Peter; Finn, Robert D.

2016-01-01

EBI metagenomics (https://www.ebi.ac.uk/metagenomics/) is a freely available hub for the analysis and archiving of metagenomic and metatranscriptomic data. Over the last 2 years, the resource has undergone rapid growth, with an increase of over five-fold in the number of processed samples and consequently represents one of the largest resources of analysed shotgun metagenomes. Here, we report the status of the resource in 2016 and give an overview of new developments. In particular, we describe updates to data content, a complete overhaul of the analysis pipeline, streamlining of data presentation via the website and the development of a new web based tool to compare functional analyses of sequence runs within a study. We also highlight two of the higher profile projects that have been analysed using the resource in the last year: the oceanographic projects Ocean Sampling Day and Tara Oceans. PMID:26582919

From gunstore to smoking gun: tracking guns that kill children in North Carolina.

PubMed

Campbell, Brendan T; Radisch, Deborah L; Phillips, J Duncan; von Allmen, Daniel

2004-12-01

This study reviews the epidemiology of pediatric firearm deaths in North Carolina and estimates the time from the retail sale of guns to their involvement in pediatric firearm deaths. The authors reviewed autopsy reports for all children 0 to 14 years of age that died of firearm-related injuries in North Carolina from January 1999 through December 2002. Data obtained included demographic information, firearm type, and manner of death. Data from the Bureau of Alcohol, Tobacco and Firearms, which traced guns involved in crimes and determined the time elapsed from purchase to their involvement in a crime (ie, time-to-crime were also reviewed). During the study period, 40 children died of firearm injuries. Mean age was 7.6 years. Handguns were responsible for the majority of deaths (59%) followed by shotguns (27%), rifles (10%), and undetermined cause (10%). Most deaths were homicides (67%) followed by unintentional death (18%), suicide (13%), and undetermined cause (2%). Most crime guns (76%) were purchased legally, and many (40%) had a time-to-crime of less than 3 years. Legally purchased firearms pose a significant threat to children in North Carolina. A more restrictive approach to the sale of handguns is a logical approach to reducing pediatric firearm-related deaths in the United States.

Nanoliter-Scale Oil-Air-Droplet Chip-Based Single Cell Proteomic Analysis.

PubMed

Li, Zi-Yi; Huang, Min; Wang, Xiu-Kun; Zhu, Ying; Li, Jin-Song; Wong, Catherine C L; Fang, Qun

2018-04-17

Single cell proteomic analysis provides crucial information on cellular heterogeneity in biological systems. Herein, we describe a nanoliter-scale oil-air-droplet (OAD) chip for achieving multistep complex sample pretreatment and injection for single cell proteomic analysis in the shotgun mode. By using miniaturized stationary droplet microreaction and manipulation techniques, our system allows all sample pretreatment and injection procedures to be performed in a nanoliter-scale droplet with minimum sample loss and a high sample injection efficiency (>99%), thus substantially increasing the analytical sensitivity for single cell samples. We applied the present system in the proteomic analysis of 100 ± 10, 50 ± 5, 10, and 1 HeLa cell(s), and protein IDs of 1360, 612, 192, and 51 were identified, respectively. The OAD chip-based system was further applied in single mouse oocyte analysis, with 355 protein IDs identified at the single oocyte level, which demonstrated its special advantages of high enrichment of sequence coverage, hydrophobic proteins, and enzymatic digestion efficiency over the traditional in-tube system.

Davenport, William (1782-1823)

NASA Astrophysics Data System (ADS)

Murdin, P.

2000-11-01

Irish astronomer, born in Dublin, became the second director of the Armagh Observatory. During eight fruitless years of idleness and decline he contributed practically nothing to astronomy until he committed suicide with a shotgun in the study of the observatory....

Microbial succession and the functional potential during the fermentation of Chinese soy sauce brine

PubMed Central

Sulaiman, Joanita; Gan, Han Ming; Yin, Wai-Fong; Chan, Kok-Gan

2014-01-01

The quality of traditional Chinese soy sauce is determined by microbial communities and their inter-related metabolic roles in the fermentation tank. In this study, traditional Chinese soy sauce brine samples were obtained periodically to monitor the transitions of the microbial population and functional properties during the 6 months of fermentation process. Whole genome shotgun method revealed that the fermentation brine was dominated by the bacterial genus Weissella and later dominated by the fungal genus Candida. Metabolic reconstruction of the metagenome sequences demonstrated a characteristic profile of heterotrophic fermentation of proteins and carbohydrates. This was supported by the detection of ethanol with stable decrease of pH values. To the best of our knowledge, this is the first study that explores the temporal changes in microbial successions over a period of 6 months, through metagenome shotgun sequencing in traditional Chinese soy sauce fermentation and the biological processes therein. PMID:25400624

Microbial succession and the functional potential during the fermentation of Chinese soy sauce brine.

PubMed

Sulaiman, Joanita; Gan, Han Ming; Yin, Wai-Fong; Chan, Kok-Gan

2014-01-01

The quality of traditional Chinese soy sauce is determined by microbial communities and their inter-related metabolic roles in the fermentation tank. In this study, traditional Chinese soy sauce brine samples were obtained periodically to monitor the transitions of the microbial population and functional properties during the 6 months of fermentation process. Whole genome shotgun method revealed that the fermentation brine was dominated by the bacterial genus Weissella and later dominated by the fungal genus Candida. Metabolic reconstruction of the metagenome sequences demonstrated a characteristic profile of heterotrophic fermentation of proteins and carbohydrates. This was supported by the detection of ethanol with stable decrease of pH values. To the best of our knowledge, this is the first study that explores the temporal changes in microbial successions over a period of 6 months, through metagenome shotgun sequencing in traditional Chinese soy sauce fermentation and the biological processes therein.

[Suicidal single intraoral shooting by a shotgun--risk of misinterpretation at the crime scene].

PubMed

Woźniak, Krzysztof; Pohl, Jerzy

2003-01-01

The authors presented two cases of suicidal single intraoral shooting by a shotgun. The first case relates to a victim found near the peak of Swinica in the Tatra mountains. When the circumstances could have suggested fatal fall from a height and minute, insignificant external injuries were found, the pistol found at the scene has been the most important indicator leading to the actual cause of death. The second case relates to a 38-year-old male found in this family house in a village. Severe internal cranial injury (bone fragmentation) was diagnosed at the scene. A self-made weapon was previously removed and hidden from the scene by a relative of the victim. Before regular forensic autopsy X-ray examination was conducted which revealed multiple intracranial foreign bodies of a shape of a shot. After the results of the autopsy the relative of the deceased indicated the location of the weapon.

Ordered shotgun sequencing of a 135 kb Xq25 YAC containing ANT2 and four possible genes, including three confirmed by EST matches.

PubMed Central

Chen, C N; Su, Y; Baybayan, P; Siruno, A; Nagaraja, R; Mazzarella, R; Schlessinger, D; Chen, E

1996-01-01

Ordered shotgun sequencing (OSS) has been successfully carried out with an Xq25 YAC substrate. yWXD703 DNA was subcloned into lambda phage and sequences of insert ends of the lambda subclones were used to generate a map to select a minimum tiling path of clones to be completely sequenced. The sequence of 135 038 nt contains the entire ANT2 cDNA as well as four other candidates suggested by computer-assisted analyses. One of the putative genes is homologous to a gene implicated in Graves' disease and it, ANT2 and two others are confirmed by EST matches. The results suggest that OSS can be applied to YACs in accord with earlier simulations and further indicate that the sequence of the YAC accurately reflects the sequence of uncloned human DNA. PMID:8918809

Current Challenges in Detecting Food Allergens by Shotgun and Targeted Proteomic Approaches: A Case Study on Traces of Peanut Allergens in Baked Cookies

PubMed Central

Pedreschi, Romina; Nørgaard, Jørgen; Maquet, Alain

2012-01-01

There is a need for selective and sensitive methods to detect the presence of food allergens at trace levels in highly processed food products. In this work, a combination of non-targeted and targeted proteomics approaches are used to illustrate the difficulties encountered in the detection of the major peanut allergens Ara h 1, Ara h 2 and Ara h 3 from a representative processed food matrix. Shotgun proteomics was employed for selection of the proteotypic peptides for targeted approaches via selective reaction monitoring. Peanut presence through detection of the proteotypic Ara h 3/4 peptides AHVQVVDSNGNR (m/z 432.5, 3+) and SPDIYNPQAGSLK (m/z 695.4, 2+) was confirmed and the developed method was able to detect peanut presence at trace levels (≥10 μg peanut g−1 matrix) in baked cookies. PMID:22413066

Recovery of a Medieval Brucella melitensis Genome Using Shotgun Metagenomics

PubMed Central

Kay, Gemma L.; Sergeant, Martin J.; Giuffra, Valentina; Bandiera, Pasquale; Milanese, Marco; Bramanti, Barbara

2014-01-01

ABSTRACT Shotgun metagenomics provides a powerful assumption-free approach to the recovery of pathogen genomes from contemporary and historical material. We sequenced the metagenome of a calcified nodule from the skeleton of a 14th-century middle-aged male excavated from the medieval Sardinian settlement of Geridu. We obtained 6.5-fold coverage of a Brucella melitensis genome. Sequence reads from this genome showed signatures typical of ancient or aged DNA. Despite the relatively low coverage, we were able to use information from single-nucleotide polymorphisms to place the medieval pathogen genome within a clade of B. melitensis strains that included the well-studied Ether strain and two other recent Italian isolates. We confirmed this placement using information from deletions and IS711 insertions. We conclude that metagenomics stands ready to document past and present infections, shedding light on the emergence, evolution, and spread of microbial pathogens. PMID:25028426

Application of whole genome shotgun sequencing for detection and characterization of genetically modified organisms and derived products.

PubMed

Holst-Jensen, Arne; Spilsberg, Bjørn; Arulandhu, Alfred J; Kok, Esther; Shi, Jianxin; Zel, Jana

2016-07-01

The emergence of high-throughput, massive or next-generation sequencing technologies has created a completely new foundation for molecular analyses. Various selective enrichment processes are commonly applied to facilitate detection of predefined (known) targets. Such approaches, however, inevitably introduce a bias and are prone to miss unknown targets. Here we review the application of high-throughput sequencing technologies and the preparation of fit-for-purpose whole genome shotgun sequencing libraries for the detection and characterization of genetically modified and derived products. The potential impact of these new sequencing technologies for the characterization, breeding selection, risk assessment, and traceability of genetically modified organisms and genetically modified products is yet to be fully acknowledged. The published literature is reviewed, and the prospects for future developments and use of the new sequencing technologies for these purposes are discussed.

A novel method for the photographic recovery of fingermark impressions from ammunition cases using digital imaging.

PubMed

Porter, Glenn; Ebeyan, Robert; Crumlish, Charles; Renshaw, Adrian

2015-03-01

The photographic preservation of fingermark impression evidence found on ammunition cases remains problematic due to the cylindrical shape of the deposition substrate preventing complete capture of the impression in a single image. A novel method was developed for the photographic recovery of fingermarks from curved surfaces using digital imaging. The process involves the digital construction of a complete impression image made from several different images captured from multiple camera perspectives. Fingermark impressions deposited onto 9-mm and 0.22-caliber brass cartridge cases and a plastic 12-gauge shotgun shell were tested using various image parameters, including digital stitching method, number of images per 360° rotation of shell, image cropping, and overlap. The results suggest that this method may be successfully used to recover fingermark impression evidence from the surfaces of ammunition cases or other similar cylindrical surfaces. © 2014 American Academy of Forensic Sciences.

A study of gunshot suicides in Northern Ireland from 1989 to 1993.

PubMed

Armour, A

1996-01-01

A study of 104 gunshot suicides, including six women, in Northern Ireland over a 5-year period. Forty-five suicides in the security forces are compared with 59 which took place in the civilian population. The former were commonly associated with marital problems and overwhelmingly occurred in young males under the age of 40, whereas the civilian deaths were predominantly associated with mental ill health, with a wider age range distribution. The security forces used rifled weapons in 44 cases, whereas civilians used shotguns in 46 cases. Twelve out of the 45 were witnessed, compared to one in the civilian population. The security forces favoured the head as site of entry in 40 cases compared to 35 in the civilian population. Alcohol consumption was involved in 23 of the security forces suicides and 18 civilian. Of the 6 women, one was in the security forces and 4 had a history of mental illness.

Diversity of thermophiles in a Malaysian hot spring determined using 16S rRNA and shotgun metagenome sequencing

PubMed Central

Chan, Chia Sing; Chan, Kok-Gan; Tay, Yea-Ling; Chua, Yi-Heng; Goh, Kian Mau

2015-01-01

The Sungai Klah (SK) hot spring is the second hottest geothermal spring in Malaysia. This hot spring is a shallow, 150-m-long, fast-flowing stream, with temperatures varying from 50 to 110°C and a pH range of 7.0–9.0. Hidden within a wooded area, the SK hot spring is continually fed by plant litter, resulting in a relatively high degree of total organic content (TOC). In this study, a sample taken from the middle of the stream was analyzed at the 16S rRNA V3-V4 region by amplicon metagenome sequencing. Over 35 phyla were detected by analyzing the 16S rRNA data. Firmicutes and Proteobacteria represented approximately 57% of the microbiome. Approximately 70% of the detected thermophiles were strict anaerobes; however, Hydrogenobacter spp., obligate chemolithotrophic thermophiles, represented one of the major taxa. Several thermophilic photosynthetic microorganisms and acidothermophiles were also detected. Most of the phyla identified by 16S rRNA were also found using the shotgun metagenome approaches. The carbon, sulfur, and nitrogen metabolism within the SK hot spring community were evaluated by shotgun metagenome sequencing, and the data revealed diversity in terms of metabolic activity and dynamics. This hot spring has a rich diversified phylogenetic community partly due to its natural environment (plant litter, high TOC, and a shallow stream) and geochemical parameters (broad temperature and pH range). It is speculated that symbiotic relationships occur between the members of the community. PMID:25798135

78 FR 84 - In the Matter of: Boniface U. Ibe, 11202 Trevor Court, Mitchellville, MD 20721

Federal Register 2010, 2011, 2012, 2013, 2014

2013-01-02

... to Nigeria, defense articles included on the United States Munitions List, namely, firearms and..., causing to exported, and attempting to export from the United States to Nigeria shotguns without having...

Proteomic analysis indicates massive changes in metabolism prior to the inhibition of growth and photosynthesis of grapevine (Vitis vinifera L.) in response to water deficit

PubMed Central

2013-01-01

Background Cabernet Sauvignon grapevines were exposed to a progressive, increasing water defict over 16 days. Shoot elongation and photosynthesis were measured for physiological responses to water deficit. The effect of water deficit over time on the abundance of individual proteins in growing shoot tips (including four immature leaves) was analyzed using nanoflow liquid chromatography - tandem mass spectrometry (nanoLC-MS/MS). Results Water deficit progressively decreased shoot elongation, stomatal conductance and photosynthesis after Day 4; 2277 proteins were identified by shotgun proteomics with an average CV of 9% for the protein abundance of all proteins. There were 472 out of 942 (50%) proteins found in all samples that were significantly affected by water deficit. The 472 proteins were clustered into four groups: increased and decreased abundance of early- and late-responding protein profiles. Vines sensed the water deficit early, appearing to acclimate to stress, because the abundance of many proteins changed before decreases in shoot elongation, stomatal conductance and photosynthesis. Predominant functional categories of the early-responding proteins included photosynthesis, glycolysis, translation, antioxidant defense and growth-related categories (steroid metabolism and water transport), whereas additional proteins for late-responding proteins were largely involved with transport, photorespiration, antioxidants, amino acid and carbohydrate metabolism. Conclusions Proteomic responses to water deficit were dynamic with early, significant changes in abundance of proteins involved in translation, energy, antioxidant defense and steroid metabolism. The abundance of these proteins changed prior to any detectable decreases in shoot elongation, stomatal conductance or photosynthesis. Many of these early-responding proteins are known to be regulated by post-transcriptional modifications such as phosphorylation. The proteomics analysis indicates massive and substantial changes in plant metabolism that appear to funnel carbon and energy into antioxidant defenses in the very early stages of plant response to water deficit before any significant injury. PMID:23514573

Understanding the gut-kidney axis in nephrolithiasis: an analysis of the gut microbiota composition and functionality of stone formers.

PubMed

Ticinesi, Andrea; Milani, Christian; Guerra, Angela; Allegri, Franca; Lauretani, Fulvio; Nouvenne, Antonio; Mancabelli, Leonardo; Lugli, Gabriele Andrea; Turroni, Francesca; Duranti, Sabrina; Mangifesta, Marta; Viappiani, Alice; Ferrario, Chiara; Dodi, Rossella; Dall'Asta, Margherita; Del Rio, Daniele; Ventura, Marco; Meschi, Tiziana

2018-04-28

The involvement of the gut microbiota in the pathogenesis of calcium nephrolithiasis has been hypothesised since the discovery of the oxalate-degrading activity of Oxalobacter formigenes , but never comprehensively studied with metagenomics. The aim of this case-control study was to compare the faecal microbiota composition and functionality between recurrent idiopathic calcium stone formers (SFs) and controls. Faecal samples were collected from 52 SFs and 48 controls (mean age 48±11). The microbiota composition was analysed through 16S rRNA microbial profiling approach. Ten samples (five SFs, five controls) were also analysed with deep shotgun metagenomics sequencing, with focus on oxalate-degrading microbial metabolic pathways. Dietary habits, assessed through a food-frequency questionnaire, and 24-hour urinary excretion of prolithogenic and antilithogenic factors, including calcium and oxalate, were compared between SFs and controls, and considered as covariates in the comparison of microbiota profiles. SFs exhibited lower faecal microbial diversity than controls (Chao1 index 1460±363vs 1658±297, fully adjusted p=0.02 with stepwise backward regression analysis). At multivariate analyses, three taxa ( Faecalibacterium , Enterobacter , Dorea ) were significantly less represented in faecal samples of SFs. The Oxalobacter abundance was not different between groups. Faecal samples from SFs exhibited a significantly lower bacterial representation of genes involved in oxalate degradation, with inverse correlation with 24-hour oxalate excretion (r=-0.87, p=0.002). The oxalate-degrading genes were represented in several bacterial species, whose cumulative abundance was inversely correlated with oxaluria (r=-0.85, p=0.02). Idiopathic calcium SFs exhibited altered gut microbiota composition and functionality that could contribute to nephrolithiasis physiopathology. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Quantitative proteomic analysis of two different rice varieties reveals that drought tolerance is correlated with reduced abundance of photosynthetic machinery and increased abundance of ClpD1 protease.

PubMed

Wu, Yunqi; Mirzaei, Mehdi; Pascovici, Dana; Chick, Joel M; Atwell, Brian J; Haynes, Paul A

2016-06-30

Rice is the major staple food for more than half of world's population. As global climate changes, we are observing more floods, droughts and severe heat waves. Two rice cultivars with contrasting genetic backgrounds and levels of tolerance to drought, Nipponbare and IAC1131, were used in this study. Four-week-old seedlings of both cultivars were grown in large soil volumes and then exposed to moderate and extreme drought for 7days, followed by 3days of re-watering. Mature leaves were harvested from plants from each treatment for protein extraction and subsequent shotgun proteomic analysis, with validation of selected proteins by western blotting. Gene Ontology (GO) annotations of differentially expressed proteins provide insights into the metabolic pathways that are involved in drought stress resistance. Our data indicate that IAC1131 appears to be better able to cope with stressful conditions by upregulating a suite of stress and defence response related proteins. Nipponbare, in contrast, lacks the range of stress responses shown by the more stress tolerant variety, and responds to drought stress by initiating a partial shutdown of chlorophyll biosynthesis in an apparent attempt to preserve resources. In this study, two rice genotypes with contrasting drought tolerance were exposed to soil water deficits, and proteomic changes were observed in mature leaf laminae. Plants were well watered and then switched to conditions of either moderate drought or extreme drought followed by three days of recovery. Proteins were identified and quantified using both label-free and Tandem Mass Tag multiplexing approaches. Several biochemical pathways were significantly altered in response to water deficit. Most notably, the up-regulation of ClpD1 protease responded strongly in the drought-tolerant landrace; this protein is typically involved in heat and osmotic stress response. In contrast, porphyrin and chlorophyll biosynthesis pathways were down-regulated, indicating suppression of the photosynthetic machinery. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparative Genome Analysis of Wheat Blue Dwarf Phytoplasma, an Obligate Pathogen That Causes Wheat Blue Dwarf Disease in China

PubMed Central

Chen, Wang; Li, Yan; Wang, Qiang; Wang, Nan; Wu, Yunfeng

2014-01-01

Wheat blue dwarf (WBD) disease is an important disease that has caused heavy losses in wheat production in northwestern China. This disease is caused by WBD phytoplasma, which is transmitted by Psammotettix striatus. Until now, no genome information about WBD phytoplasma has been published, seriously restricting research on this obligate pathogen. In this paper, we report a new sequencing and assembling strategy for phytoplasma genome projects. This strategy involves differential centrifugation, pulsed-field gel electrophoresis, whole genome amplification, shotgun sequencing, de novo assembly, screening of contigs from phytoplasma and the connection of phytoplasma contigs. Using this scheme, the WBD phytoplasma draft genome was obtained. It was comprised of six contigs with a total size of 611,462 bp, covering ∼94% of the chromosome. Five-hundred-twenty-five protein-coding genes, two operons for rRNA genes and 32 tRNA genes were identified. Comparative genome analyses between WBD phytoplasma and other phytoplasmas were subsequently carried out. The results showed that extensive arrangements and inversions existed among the WBD, OY-M and AY-WB phytoplasma genomes. Most protein-coding genes in WBD phytoplasma were found to be homologous to genes from other phytoplasmas; only 22 WBD-specific genes were identified. KEGG pathway analysis indicated that WBD phytoplasma had strongly reduced metabolic capabilities. However, 46 transporters were identified, which were involved with dipeptides/oligopeptides, spermidine/putrescine, cobalt and Mn/Zn transport, and so on. A total of 37 secreted proteins were encoded in the WBD phytoplasma chromosome and plasmids. Of these, three secreted proteins were similar to the reported phytoplasma virulence factors TENGU, SAP11 and SAP54. In addition, WBD phytoplasma possessed several proteins that were predicted to play a role in its adaptation to diverse environments. These results will provide clues for research on the pathogenic mechanisms of WBD phytoplasma and will also provide a perspective about the genome sequencing of other phytoplasmas and obligate organisms. PMID:24798075

A Multi-Platform Draft de novo Genome Assembly and Comparative Analysis for the Scarlet Macaw (Ara macao)

PubMed Central

Seabury, Christopher M.; Dowd, Scot E.; Seabury, Paul M.; Raudsepp, Terje; Brightsmith, Donald J.; Liboriussen, Poul; Halley, Yvette; Fisher, Colleen A.; Owens, Elaine; Viswanathan, Ganesh; Tizard, Ian R.

2013-01-01

Data deposition to NCBI Genomes This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession AMXX00000000 (SMACv1.0, unscaffolded genome assembly). The version described in this paper is the first version (AMXX01000000). The scaffolded assembly (SMACv1.1) has been deposited at DDBJ/EMBL/GenBank under the accession AOUJ00000000, and is also the first version (AOUJ01000000). Strong biological interest in traits such as the acquisition and utilization of speech, cognitive abilities, and longevity catalyzed the utilization of two next-generation sequencing platforms to provide the first-draft de novo genome assembly for the large, new world parrot Ara macao (Scarlet Macaw). Despite the challenges associated with genome assembly for an outbred avian species, including 951,507 high-quality putative single nucleotide polymorphisms, the final genome assembly (>1.035 Gb) includes more than 997 Mb of unambiguous sequence data (excluding N’s). Cytogenetic analyses including ZooFISH revealed complex rearrangements associated with two scarlet macaw macrochromosomes (AMA6, AMA7), which supports the hypothesis that translocations, fusions, and intragenomic rearrangements are key factors associated with karyotype evolution among parrots. In silico annotation of the scarlet macaw genome provided robust evidence for 14,405 nuclear gene annotation models, their predicted transcripts and proteins, and a complete mitochondrial genome. Comparative analyses involving the scarlet macaw, chicken, and zebra finch genomes revealed high levels of nucleotide-based conservation as well as evidence for overall genome stability among the three highly divergent species. Application of a new whole-genome analysis of divergence involving all three species yielded prioritized candidate genes and noncoding regions for parrot traits of interest (i.e., speech, intelligence, longevity) which were independently supported by the results of previous human GWAS studies. We also observed evidence for genes and noncoding loci that displayed extreme conservation across the three avian lineages, thereby reflecting their likely biological and developmental importance among birds. PMID:23667475

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.

Toward Accurate and Quantitative Comparative Metagenomics

PubMed Central

Nayfach, Stephen; Pollard, Katherine S.

2016-01-01

Shotgun metagenomics and computational analysis are used to compare the taxonomic and functional profiles of microbial communities. Leveraging this approach to understand roles of microbes in human biology and other environments requires quantitative data summaries whose values are comparable across samples and studies. Comparability is currently hampered by the use of abundance statistics that do not estimate a meaningful parameter of the microbial community and biases introduced by experimental protocols and data-cleaning approaches. Addressing these challenges, along with improving study design, data access, metadata standardization, and analysis tools, will enable accurate comparative metagenomics. We envision a future in which microbiome studies are replicable and new metagenomes are easily and rapidly integrated with existing data. Only then can the potential of metagenomics for predictive ecological modeling, well-powered association studies, and effective microbiome medicine be fully realized. PMID:27565341

Comprehensive proteomic analysis of Penicillium verrucosum.

PubMed

Nöbauer, Katharina; Hummel, Karin; Mayrhofer, Corina; Ahrens, Maike; Setyabudi, Francis M C; Schmidt-Heydt, Markus; Eisenacher, Martin; Razzazi-Fazeli, Ebrahim

2017-05-01

Mass spectrometric identification of proteins in species lacking validated sequence information is a major problem in veterinary science. In the present study, we used ochratoxin A producing Penicillium verrucosum to identify and quantitatively analyze proteins of an organism with yet no protein information available. The work presented here aimed to provide a comprehensive protein identification of P. verrucosum using shotgun proteomics. We were able to identify 3631 proteins in an "ab initio" translated database from DNA sequences of P. verrucosum. Additionally, a sequential window acquisition of all theoretical fragment-ion spectra analysis was done to find differentially regulated proteins at two different time points of the growth curve. We compared the proteins at the beginning (day 3) and at the end of the log phase (day 12). © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Combining results of multiple search engines in proteomics.

PubMed

Shteynberg, David; Nesvizhskii, Alexey I; Moritz, Robert L; Deutsch, Eric W

2013-09-01

A crucial component of the analysis of shotgun proteomics datasets is the search engine, an algorithm that attempts to identify the peptide sequence from the parent molecular ion that produced each fragment ion spectrum in the dataset. There are many different search engines, both commercial and open source, each employing a somewhat different technique for spectrum identification. The set of high-scoring peptide-spectrum matches for a defined set of input spectra differs markedly among the various search engine results; individual engines each provide unique correct identifications among a core set of correlative identifications. This has led to the approach of combining the results from multiple search engines to achieve improved analysis of each dataset. Here we review the techniques and available software for combining the results of multiple search engines and briefly compare the relative performance of these techniques.

Combining Results of Multiple Search Engines in Proteomics*

PubMed Central

Shteynberg, David; Nesvizhskii, Alexey I.; Moritz, Robert L.; Deutsch, Eric W.

2013-01-01

A crucial component of the analysis of shotgun proteomics datasets is the search engine, an algorithm that attempts to identify the peptide sequence from the parent molecular ion that produced each fragment ion spectrum in the dataset. There are many different search engines, both commercial and open source, each employing a somewhat different technique for spectrum identification. The set of high-scoring peptide-spectrum matches for a defined set of input spectra differs markedly among the various search engine results; individual engines each provide unique correct identifications among a core set of correlative identifications. This has led to the approach of combining the results from multiple search engines to achieve improved analysis of each dataset. Here we review the techniques and available software for combining the results of multiple search engines and briefly compare the relative performance of these techniques. PMID:23720762

Toward Accurate and Quantitative Comparative Metagenomics.

PubMed

Nayfach, Stephen; Pollard, Katherine S

2016-08-25

Shotgun metagenomics and computational analysis are used to compare the taxonomic and functional profiles of microbial communities. Leveraging this approach to understand roles of microbes in human biology and other environments requires quantitative data summaries whose values are comparable across samples and studies. Comparability is currently hampered by the use of abundance statistics that do not estimate a meaningful parameter of the microbial community and biases introduced by experimental protocols and data-cleaning approaches. Addressing these challenges, along with improving study design, data access, metadata standardization, and analysis tools, will enable accurate comparative metagenomics. We envision a future in which microbiome studies are replicable and new metagenomes are easily and rapidly integrated with existing data. Only then can the potential of metagenomics for predictive ecological modeling, well-powered association studies, and effective microbiome medicine be fully realized. Copyright © 2016 Elsevier Inc. All rights reserved.

Proteomic Analysis of a NAP1 Clostridium difficile Clinical Isolate Resistant to Metronidazole

PubMed Central

Chong, Patrick M.; Lynch, Tarah; McCorrister, Stuart; Kibsey, Pamela; Miller, Mark; Gravel, Denise; Westmacott, Garrett R.; Mulvey, Michael R.

2014-01-01

Background Clostridium difficile is an anaerobic, Gram-positive bacterium that has been implicated as the leading cause of antibiotic-associated diarrhea. Metronidazole is currently the first-line treatment for mild to moderate C. difficile infections. Our laboratory isolated a strain of C. difficile with a stable resistance phenotype to metronidazole. A shotgun proteomics approach was used to compare differences in the proteomes of metronidazole-resistant and -susceptible isolates. Methodology/Principal Findings NAP1 C. difficile strains CD26A54_R (Met-resistant), CD26A54_S (reduced- susceptibility), and VLOO13 (Met-susceptible) were grown to mid-log phase, and spiked with metronidazole at concentrations 2 doubling dilutions below the MIC. Peptides from each sample were labeled with iTRAQ and subjected to 2D-LC-MS/MS analysis. In the absence of metronidazole, higher expression was observed of some proteins in C. difficile strains CD26A54_S and CD26A54_R that may be involved with reduced susceptibility or resistance to metronidazole, including DNA repair proteins, putative nitroreductases, and the ferric uptake regulator (Fur). After treatment with metronidazole, moderate increases were seen in the expression of stress-related proteins in all strains. A moderate increase was also observed in the expression of the DNA repair protein RecA in CD26A54_R. Conclusions/Significance This study provided an in-depth proteomic analysis of a stable, metronidazole-resistant C. difficile isolate. The results suggested that a multi-factorial response may be associated with high level metronidazole-resistance in C. difficile, including the possible roles of altered iron metabolism and/or DNA repair. PMID:24400070

CAFE: aCcelerated Alignment-FrEe sequence analysis.

PubMed

Lu, Yang Young; Tang, Kujin; Ren, Jie; Fuhrman, Jed A; Waterman, Michael S; Sun, Fengzhu

2017-07-03

Alignment-free genome and metagenome comparisons are increasingly important with the development of next generation sequencing (NGS) technologies. Recently developed state-of-the-art k-mer based alignment-free dissimilarity measures including CVTree, $d_2^*$ and $d_2^S$ are more computationally expensive than measures based solely on the k-mer frequencies. Here, we report a standalone software, aCcelerated Alignment-FrEe sequence analysis (CAFE), for efficient calculation of 28 alignment-free dissimilarity measures. CAFE allows for both assembled genome sequences and unassembled NGS shotgun reads as input, and wraps the output in a standard PHYLIP format. In downstream analyses, CAFE can also be used to visualize the pairwise dissimilarity measures, including dendrograms, heatmap, principal coordinate analysis and network display. CAFE serves as a general k-mer based alignment-free analysis platform for studying the relationships among genomes and metagenomes, and is freely available at https://github.com/younglululu/CAFE. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

To prohibit the Department of Justice from tracking and cataloguing the purchases of multiple rifles and shotguns.

THOMAS, 113th Congress

Rep. Schweikert, David [R-AZ-6

2013-06-18

House - 07/15/2013 Referred to the Subcommittee on Crime, Terrorism, Homeland Security, and Investigations. (All Actions) Tracker: This bill has the status IntroducedHere are the steps for Status of Legislation:

Genome sequence of Stachybotrys chartarum Strain 51-11

EPA Science Inventory

Stachybotrys chartarum strain 51-11 genome was sequenced by shotgun sequencing utilizing Illumina Hiseq 2000 and PacBio long read technology. Since Stachybotrys chartarum has been implicated in health impacts within water-damaged buildings, any information extracted from the geno...

50 CFR 21.52 - Public health control order for resident Canada geese.

Code of Federal Regulations, 2010 CFR

2010-10-01

... to human health. (c) What is a direct threat to human health? A direct threat to human health is one... use must comply with any labeling restrictions. (iv) Persons using shotguns must use nontoxic shot, as...

50 CFR 21.52 - Public health control order for resident Canada geese.

Code of Federal Regulations, 2012 CFR

2012-10-01

... to human health. (c) What is a direct threat to human health? A direct threat to human health is one... use must comply with any labeling restrictions. (iv) Persons using shotguns must use nontoxic shot, as...

50 CFR 21.52 - Public health control order for resident Canada geese.

Code of Federal Regulations, 2014 CFR

2014-10-01

... to human health. (c) What is a direct threat to human health? A direct threat to human health is one... use must comply with any labeling restrictions. (iv) Persons using shotguns must use nontoxic shot, as...

50 CFR 21.52 - Public health control order for resident Canada geese.

Code of Federal Regulations, 2013 CFR

2013-10-01

... to human health. (c) What is a direct threat to human health? A direct threat to human health is one... use must comply with any labeling restrictions. (iv) Persons using shotguns must use nontoxic shot, as...

78 FR 85 - In the Matter of: Emenike Charles Nwankwoala Currently Incarcerated at: Inmate Number 50756-037...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-01-02

... willfully exporting, causing to be exported, and attempting to export from the United States to Nigeria..., causing to exported, and attempting to export from the United States to Nigeria shotguns without having...

Phosphoproteomics of Klebsiella pneumoniae NTUH-K2044 Reveals a Tight Link between Tyrosine Phosphorylation and Virulence*

PubMed Central

Lin, Miao-Hsia; Hsu, Tung-Li; Lin, Shu-Yu; Pan, Yi-Jiun; Jan, Jia-Tsrong; Wang, Jin-Town; Khoo, Kay-Hooi; Wu, Shih-Hsiung

2009-01-01

Encapsulated Klebsiella pneumoniae is the predominant causative agent of pyogenic liver abscess, an emerging infectious disease that often complicates metastatic meningitis or endophthalmitis. The capsular polysaccharide on K. pneumoniae surface was determined as the key to virulence. Although the regulation of capsular polysaccharide biosynthesis is largely unclear, it was found that protein-tyrosine kinases and phosphatases are involved. Therefore, the identification and characterization of such kinases, phosphatases, and their substrates would advance our knowledge of the underlying mechanism in capsule formation and could contribute to the development of new therapeutic strategies. Here, we analyzed the phosphoproteome of K. pneumoniae NTUH-K2044 with a shotgun approach and identified 117 unique phosphopeptides along with 93 in vivo phosphorylated sites corresponding to 81 proteins. Interestingly, three of the identified tyrosine phosphorylated proteins, namely protein-tyrosine kinase (Wzc), phosphomannomutase (ManB), and undecaprenyl-phosphate glycosyltransferase (WcaJ), were found to be distributed in the cps locus and thus were speculated to be involved in the converging signal transduction of capsule biosynthesis. Consequently, we decided to focus on the lesser studied ManB and WcaJ for mutation analysis. The capsular polysaccharides of WcaJ mutant (WcaJY5F) were dramatically reduced quantitatively, and the LD50 increased by 200-fold in a mouse peritonitis model compared with the wild-type strain. However, the capsular polysaccharides of ManB mutant (ManBY26F) showed no difference in quantity, and the LD50 increased by merely 6-fold in mice test. Our study provided a clear trend that WcaJ tyrosine phosphorylation can regulate the biosynthesis of capsular polysaccharides and result in the pathogenicity of K. pneumoniae NTUH-K2044. PMID:19696081

Metagenomics reveals sediment microbial community response to Deepwater Horizon oil spill

PubMed Central

Mason, Olivia U; Scott, Nicole M; Gonzalez, Antonio; Robbins-Pianka, Adam; Bælum, Jacob; Kimbrel, Jeffrey; Bouskill, Nicholas J; Prestat, Emmanuel; Borglin, Sharon; Joyner, Dominique C; Fortney, Julian L; Jurelevicius, Diogo; Stringfellow, William T; Alvarez-Cohen, Lisa; Hazen, Terry C; Knight, Rob; Gilbert, Jack A; Jansson, Janet K

2014-01-01

The Deepwater Horizon (DWH) oil spill in the spring of 2010 resulted in an input of ∼4.1 million barrels of oil to the Gulf of Mexico; >22% of this oil is unaccounted for, with unknown environmental consequences. Here we investigated the impact of oil deposition on microbial communities in surface sediments collected at 64 sites by targeted sequencing of 16S rRNA genes, shotgun metagenomic sequencing of 14 of these samples and mineralization experiments using 14C-labeled model substrates. The 16S rRNA gene data indicated that the most heavily oil-impacted sediments were enriched in an uncultured Gammaproteobacterium and a Colwellia species, both of which were highly similar to sequences in the DWH deep-sea hydrocarbon plume. The primary drivers in structuring the microbial community were nitrogen and hydrocarbons. Annotation of unassembled metagenomic data revealed the most abundant hydrocarbon degradation pathway encoded genes involved in degrading aliphatic and simple aromatics via butane monooxygenase. The activity of key hydrocarbon degradation pathways by sediment microbes was confirmed by determining the mineralization of 14C-labeled model substrates in the following order: propylene glycol, dodecane, toluene and phenanthrene. Further, analysis of metagenomic sequence data revealed an increase in abundance of genes involved in denitrification pathways in samples that exceeded the Environmental Protection Agency (EPA)'s benchmarks for polycyclic aromatic hydrocarbons (PAHs) compared with those that did not. Importantly, these data demonstrate that the indigenous sediment microbiota contributed an important ecosystem service for remediation of oil in the Gulf. However, PAHs were more recalcitrant to degradation, and their persistence could have deleterious impacts on the sediment ecosystem. PMID:24451203

Metagenomics reveals sediment microbial community response to Deepwater Horizon oil spill

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

Mason, Olivia U.; Scott, Nicole M.; Gonzalez, Antonio

The Deepwater Horizon (DWH) oil spill in the spring of 2010 resulted in an input of ~4.1 million barrels of oil to the Gulf of Mexico; >22% of this oil is unaccounted for, with unknown environmental consequences. Here we investigated the impact of oil deposition on microbial communities in surface sediments collected at 64 sites by targeted sequencing of 16S rRNA genes, shotgun metagenomic sequencing of 14 of these samples and mineralization experiments using 14C-labeled model substrates. The 16S rRNA gene data indicated that the most heavily oil-impacted sediments were enriched in an uncultured Gammaproteobacterium and a Colwellia species, bothmore » of which were highly similar to sequences in the DWH deep-sea hydrocarbon plume. The primary drivers in structuring the microbial community were nitrogen and hydrocarbons. Annotation of unassembled metagenomic data revealed the most abundant hydrocarbon degradation pathway encoded genes involved in degrading aliphatic and simple aromatics via butane monooxygenase. The activity of key hydrocarbon degradation pathways by sediment microbes was confirmed by determining the mineralization of 14C-labeled model substrates in the following order: propylene glycol, dodecane, toluene and phenanthrene. Further, analysis of metagenomic sequence data revealed an increase in abundance of genes involved in denitrification pathways in samples that exceeded the Environmental Protection Agency (EPA)’s benchmarks for polycyclic aromatic hydrocarbons (PAHs) compared with those that did not. Importantly, these data demonstrate that the indigenous sediment microbiota contributed an important ecosystem service for remediation of oil in the Gulf. However, PAHs were more recalcitrant to degradation, and their persistence could have deleterious impacts on the sediment ecosystem.« less

Urinary microbiome of kidney transplant patients reveals dysbiosis with potential for antibiotic resistance

PubMed Central

Rani, Asha; Ranjan, Ravi; McGee, Halvor S.; Andropolis, Kalista E.; Panchal, Dipti V.; Hajjiri, Zahraa; Brennan, Daniel C.; Finn, Patricia W.; Perkins, David L.

2016-01-01

Recent studies have established that a complex community of microbes colonize the human urinary tract; however their role in kidney transplant patients treated with prophylactic antibiotics remains poorly investigated. Our aim was to investigate the urinary microbiome of kidney transplant recipients. Urine samples from 21 patients following kidney transplantation and 8 healthy controls, were collected. All patients received prophylactic treatment with the antibiotic trimethoprim/sulfamethoxazole. Metagenomic DNA was isolated from urine samples, sequenced using metagenomics shotgun sequencing approach on Illumina HiSeq2000 platform, and analyzed for microbial taxonomic and functional annotations. Our results demonstrate that the urine microbiome of kidney transplants was markedly different at all taxonomic levels from phyla to species, had decreased microbial diversity and increased abundance of potentially pathogenic species compared to healthy controls. Specifically, at the phylum level we detected a significant decrease in Actinobacteria and increase in Firmicutes due to increases in Enterococcus faecalis. In addition, there was an increase in the Proteobacteria due to increases in E. coli. Analysis of predicted functions of the urinary metagenome revealed increased abundance of enzymes in the folate pathway including dihydrofolate synthase that are not inhibited by trimethoprim/sulfamethoxazole, but can augment folate metabolism. This report characterizes the urinary microbiome of kidney transplants using shotgun metagenomics approach. Our results indicate that the urinary microbiota may be modified in the context of prophylactic antibiotics, indicating that a therapeutic intervention may shift the urinary microbiota to select bacterial species with increased resistance to antibiotics. The evaluation and development of optimal prophylactic regimens that do not promote antibiotic resistance is an important future goal. PMID:27669488

Breakdown of Phylogenetic Signal: A Survey of Microsatellite Densities in 454 Shotgun Sequences from 154 Non Model Eukaryote Species

PubMed Central

Meglécz, Emese; Nève, Gabriel; Biffin, Ed; Gardner, Michael G.

2012-01-01

Microsatellites are ubiquitous in Eukaryotic genomes. A more complete understanding of their origin and spread can be gained from a comparison of their distribution within a phylogenetic context. Although information for model species is accumulating rapidly, it is insufficient due to a lack of species depth, thus intragroup variation is necessarily ignored. As such, apparent differences between groups may be overinflated and generalizations cannot be inferred until an analysis of the variation that exists within groups has been conducted. In this study, we examined microsatellite coverage and motif patterns from 454 shotgun sequences of 154 Eukaryote species from eight distantly related phyla (Cnidaria, Arthropoda, Onychophora, Bryozoa, Mollusca, Echinodermata, Chordata and Streptophyta) to test if a consistent phylogenetic pattern emerges from the microsatellite composition of these species. It is clear from our results that data from model species provide incomplete information regarding the existing microsatellite variability within the Eukaryotes. A very strong heterogeneity of microsatellite composition was found within most phyla, classes and even orders. Autocorrelation analyses indicated that while microsatellite contents of species within clades more recent than 200 Mya tend to be similar, the autocorrelation breaks down and becomes negative or non-significant with increasing divergence time. Therefore, the age of the taxon seems to be a primary factor in degrading the phylogenetic pattern present among related groups. The most recent classes or orders of Chordates still retain the pattern of their common ancestor. However, within older groups, such as classes of Arthropods, the phylogenetic pattern has been scrambled by the long independent evolution of the lineages. PMID:22815847

Modified filter-aided sample preparation (FASP) method increases peptide and protein identifications for shotgun proteomics.

PubMed

Ni, Mao-Wei; Wang, Lu; Chen, Wei; Mou, Han-Zhou; Zhou, Jie; Zheng, Zhi-Guo

2017-01-30

Mass spectrometry (MS)-based protein identification depends mainly on protein extraction and digestion. Although sodium dodecyl sulfate (SDS) can preclude enzymatic digestion and interfere with MS analysis, it is still the most widely used surfactant in these steps. To overcome these disadvantages, a SDS-compatible proteomic technique for SDS removal prior to MS-based analyses was developed, namely filter-aided sample preparation (FASP). Herein, based on the effectiveness of sodium deoxycholate and a detergent removal spin column, we developed a modified FASP (mFASP) method and compared its overall performance, total number of peptides and proteins identified for shotgun proteomic experiments with that of the FASP method. Identification of 4570 ± 392 and 9139 ± 317 peptides and description of 862 ± 46 and 1377 ± 33 protein groups with two or more peptides from the ovarian cancer cell line A2780 was accomplished by FASP and mFASP methods, respectively. The mFASP method (21.2 ± 0.2%) had higher average peptide to protein coverage than FASP method (13.2 ± 0.5%). More hydrophobic peptides were identified by mFASP than by FASP, as indicated by the GRAVY score distribution. The reported method enables reliable and efficient identification of proteins and peptides in whole-cell extracts containing SDS. The new approach allows for higher throughput (the simultaneous identification of more proteins), a more comprehensive investigation of proteins, and potentially the discovery of new biomarkers. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Pulling out the 1%: Whole-Genome Capture for the Targeted Enrichment of Ancient DNA Sequencing Libraries

PubMed Central

Carpenter, Meredith L.; Buenrostro, Jason D.; Valdiosera, Cristina; Schroeder, Hannes; Allentoft, Morten E.; Sikora, Martin; Rasmussen, Morten; Gravel, Simon; Guillén, Sonia; Nekhrizov, Georgi; Leshtakov, Krasimir; Dimitrova, Diana; Theodossiev, Nikola; Pettener, Davide; Luiselli, Donata; Sandoval, Karla; Moreno-Estrada, Andrés; Li, Yingrui; Wang, Jun; Gilbert, M. Thomas P.; Willerslev, Eske; Greenleaf, William J.; Bustamante, Carlos D.

2013-01-01

Most ancient specimens contain very low levels of endogenous DNA, precluding the shotgun sequencing of many interesting samples because of cost. Ancient DNA (aDNA) libraries often contain <1% endogenous DNA, with the majority of sequencing capacity taken up by environmental DNA. Here we present a capture-based method for enriching the endogenous component of aDNA sequencing libraries. By using biotinylated RNA baits transcribed from genomic DNA libraries, we are able to capture DNA fragments from across the human genome. We demonstrate this method on libraries created from four Iron Age and Bronze Age human teeth from Bulgaria, as well as bone samples from seven Peruvian mummies and a Bronze Age hair sample from Denmark. Prior to capture, shotgun sequencing of these libraries yielded an average of 1.2% of reads mapping to the human genome (including duplicates). After capture, this fraction increased substantially, with up to 59% of reads mapped to human and enrichment ranging from 6- to 159-fold. Furthermore, we maintained coverage of the majority of regions sequenced in the precapture library. Intersection with the 1000 Genomes Project reference panel yielded an average of 50,723 SNPs (range 3,062–147,243) for the postcapture libraries sequenced with 1 million reads, compared with 13,280 SNPs (range 217–73,266) for the precapture libraries, increasing resolution in population genetic analyses. Our whole-genome capture approach makes it less costly to sequence aDNA from specimens containing very low levels of endogenous DNA, enabling the analysis of larger numbers of samples. PMID:24568772

P7-S Combining Workflow-Based Project Organization with Protein-Dependant Data Retrieval for the Retrieval of Extensive Proteome Information

PubMed Central

Glandorf, J.; Thiele, H.; Macht, M.; Vorm, O.; Podtelejnikov, A.

2007-01-01

In the course of a full-scale proteomics experiment, the handling of the data as well as the retrieval of the relevant information from the results is a major challenge due to the massive amount of generated data (gel images, chromatograms, and spectra) as well as associated result information (sequences, literature, etc.). To obtain meaningful information from these data, one has to filter the results in an easy way. Possibilities to do so can be based on GO terms or structural features such as transmembrane domains, involvement in certain pathways, etc. In this presentation we will show how a combination of a software package with a workflow-based result organization (Bruker ProteinScape) and a protein-centered data-mining software (Proxeon ProteinCenter) can assist in the comparison of the results from large projects, such as comparison of cross-platform results from 2D PAGE/MS with shotgun LC-ESI-MS/MS. We will present differences between different technologies and show how these differences can be easily identified and how they allow us to draw conclusions on the involved technologies.

Proteomic insights into the protective mechanisms of an in vitro oxidative stress model of early stage Parkinson's disease.

PubMed

Bauereis, Brian; Haskins, William E; Lebaron, Richard G; Renthal, Robert

2011-01-13

Previous studies in Parkinson's disease (PD) models suggest that early events along the path to neurodegeneration involve activation of the ubiquitin-proteasome system (UPS), endoplasmic reticulum-associated degradation (ERAD), and the unfolded protein response (UPR) pathways, in both the sporadic and familial forms of the disease, and thus ER stress may be a common feature. Furthermore, impairments in protein degradation have been linked to oxidative stress as well as pathways associated with ER stress. We hypothesize that oxidative stress is a primary initiator in a multi-factorial cascade driving dopaminergic (DA) neurons towards death in the early stages of the disease. We now report results from proteomic analysis of a rotenone-induced oxidative stress model of PD in the human neuroblastoma cell line, SH-SY5Y. Cells were exposed to sub-micromolar concentrations of rotenone for 48h prior to whole cell protein extraction and shotgun proteomic analysis. Evidence for activation of the UPR comes from our observation of up-regulated binding immunoglobulin protein (BiP), heat shock proteins, and foldases. We also observed up-regulation of proteins that contribute to the degradation of misfolded or unfolded proteins controlled by the UPS and ERAD pathways. Activation of the UPR may allow neurons to maintain protein homeostasis in the cytosol and ER despite an increase in reactive oxygen species due to oxidative stress, and activation of the UPS and ERAD may further augment clean-up and quality control in the cell. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

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

Takada, Michiya; Ban, Yoshiyuki, E-mail: yshyban@yahoo.co.jp; Yamamoto, Gou

Research highlights: {yields} In proliferative membrane and epiretinal membrane specimens, the numbers of proteins are 225 and 154, respectively, and 123 proteins are common to both. {yields} Periostin and thrombospondin-1 proteins are unique to the proliferative membrane specimens. {yields} The expression of periostin is significantly up-regulated in proliferative membrane specimens. -- Abstract: Diabetes can lead to serious microvascular complications including proliferative diabetic retinopathy (PDR), the leading cause of blindness in adults. Recent studies using gene array technology have attempted to apply a hypothesis-generating approach to elucidate the pathogenesis of PDR, but these studies rely on mRNA differences, which may ormore » may not be related to significant biological processes. To better understand the basic mechanisms of PDR and to identify potential new biomarkers, we performed shotgun liquid chromatography (LC)/tandem mass spectrometry (MS/MS) analysis on pooled protein extracts from neovascular membranes obtained from PDR specimens and compared the results with those from non-vascular epiretinal membrane (ERM) specimens. We detected 226 distinct proteins in neovascular membranes and 154 in ERM. Among these proteins, 102 were specific to neovascular membranes and 30 were specific to ERM. We identified a candidate marker, periostin, as well as several known PDR markers such as pigment epithelium-derived factor (PEDF). We then performed RT-PCR using these markers. The expression of periostin was significantly up-regulated in proliferative membrane specimens. Periostin induces cell attachment and spreading and plays a role in cell adhesion. Proteomic analysis by LC/MS/MS, which permits accurate quantitative comparison, was useful in identifying new candidates such as periostin potentially involved in the pathogenesis of PDR.« less

Analysis of the Cerebrospinal Fluid Proteome in Alzheimer's Disease

PubMed Central

Khoonsari, Payam Emami; Häggmark, Anna; Lönnberg, Maria; Mikus, Maria; Kilander, Lena; Lannfelt, Lars; Bergquist, Jonas; Ingelsson, Martin; Nilsson, Peter

2016-01-01

Alzheimer’s disease is a neurodegenerative disorder accounting for more than 50% of cases of dementia. Diagnosis of Alzheimer’s disease relies on cognitive tests and analysis of amyloid beta, protein tau, and hyperphosphorylated tau in cerebrospinal fluid. Although these markers provide relatively high sensitivity and specificity for early disease detection, they are not suitable for monitor of disease progression. In the present study, we used label-free shotgun mass spectrometry to analyse the cerebrospinal fluid proteome of Alzheimer’s disease patients and non-demented controls to identify potential biomarkers for Alzheimer’s disease. We processed the data using five programs (DecyderMS, Maxquant, OpenMS, PEAKS, and Sieve) and compared their results by means of reproducibility and peptide identification, including three different normalization methods. After depletion of high abundant proteins we found that Alzheimer’s disease patients had lower fraction of low-abundance proteins in cerebrospinal fluid compared to healthy controls (p<0.05). Consequently, global normalization was found to be less accurate compared to using spiked-in chicken ovalbumin for normalization. In addition, we determined that Sieve and OpenMS resulted in the highest reproducibility and PEAKS was the programs with the highest identification performance. Finally, we successfully verified significantly lower levels (p<0.05) of eight proteins (A2GL, APOM, C1QB, C1QC, C1S, FBLN3, PTPRZ, and SEZ6) in Alzheimer’s disease compared to controls using an antibody-based detection method. These proteins are involved in different biological roles spanning from cell adhesion and migration, to regulation of the synapse and the immune system. PMID:26950848

Genome Sequencing and Analysis of the Biomass-Degrading Fungus Trichoderma reesei (syn. Hypocrea jecorina)

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

Martinez, Antonio D.; Berka, Randy; Henrissat, Bernard

2008-05-01

A major thrust of the white biotechnology movement involves the development of enzyme systems which depolymerize biomass to simple sugars which are subsequently converted to sustainable biofuels (e.g., ethanol) and chemical intermediates. The fungus Trichoderma reesei (syn. Hypocrea jecorina) represents a paradigm for the industrial production of highly efficient cellulases and hemicellulases needed for hydrolysis of biomass polysaccharides. Herein we describe intriguing attributes of the T. reeseigenome in relation to the future of fuel biotechnology. The T. reesei genome sequence was derived using a whole genome shotgun approach combined with finishing work to generate an assembly comprising 89 scaffolds totalingmore » 34 Mbp with few gaps. In total, 9,130 gene models were predicted using a combination of ab initio and sequence similarity-based methods and EST data. Considering the industrial utility and effectiveness of its enzymes, the T. reesei genome surprisingly encodes the fewest cellulases and hemicellulases of any fungus having the ability to hydrolyze plant cell wall polysaccharides and whose genome has been sequenced. Many genes encoding carbohydrate active enzymes are distributed non-randomly in groups or clusters that interestingly lie between regions of synteny with other Sordariomycetes. Additionally, the T. reesei genome contains a multitude of genes encoding biosynthetic pathways for secondary metabolites (possible antibacterial and antifungal compounds) which may promote successful competition and survival in the crowded and competitive soil habitat occupied by T. reesei. Our analysis coupled with the availability of genome sequence data provides a roadmap for construction of enhanced T. reesei strains for industrial applications.« less

Quantitative Proteomic Analysis of Wheat Cultivars with Differing Drought Stress Tolerance

PubMed Central

Ford, Kristina L.; Cassin, Andrew; Bacic, Antony

2011-01-01

Using a series of multiplexed experiments we studied the quantitative changes in protein abundance of three Australian bread wheat cultivars (Triticum aestivum L.) in response to a drought stress. Three cultivars differing in their ability to maintain grain yield during drought, Kukri (intolerant), Excalibur (tolerant), and RAC875 (tolerant), were grown in the glasshouse with cyclic drought treatment that mimicked conditions in the field. Proteins were isolated from leaves of mature plants and isobaric tags were used to follow changes in the relative protein abundance of 159 proteins. This is the first shotgun proteomics study in wheat, providing important insights into protein responses to drought as well as identifying the largest number of wheat proteins (1,299) in a single study. The changes in the three cultivars at the different time points reflected their differing physiological responses to drought, with the two drought tolerant varieties (Excalibur and RAC875) differing in their protein responses. Excalibur lacked significant changes in proteins during the initial onset of the water deficit in contrast to RAC875 that had a large number of significant changes. All three cultivars had changes consistent with an increase in oxidative stress metabolism and reactive O2 species (ROS) scavenging capacity seen through increases in superoxide dismutases and catalases as well as ROS avoidance through the decreases in proteins involved in photosynthesis and the Calvin cycle. PMID:22639595

Shotgun Metagenomic Profiles Have a High Capacity To Discriminate Samples of Activated Sludge According to Wastewater Type

PubMed Central

Ibarbalz, Federico M.; Orellana, Esteban; Figuerola, Eva L. M.

2016-01-01

ABSTRACT This study was conducted to investigate whether functions encoded in the metagenome could improve our ability to understand the link between microbial community structures and functions in activated sludge. By analyzing data sets from six industrial and six municipal wastewater treatment plants (WWTPs), covering different configurations, operational conditions, and geographic regions, we found that wastewater influent composition was an overriding factor shaping the metagenomic composition of the activated sludge samples. Community GC content profiles were conserved within treatment plants on a time scale of years and between treatment plants with similar influent wastewater types. Interestingly, GC contents of the represented phyla covaried with the average GC contents of the corresponding WWTP metagenome. This suggests that the factors influencing nucleotide composition act similarly across taxa and thus the variation in nucleotide contents is driven by environmental differences between WWTPs. While taxonomic richness and functional richness were correlated, shotgun metagenomics complemented taxon-based analyses in the task of classifying microbial communities involved in wastewater treatment systems. The observed taxonomic dissimilarity between full-scale WWTPs receiving influent types with varied compositions, as well as the inferred taxonomic and functional assignment of recovered genomes from each metagenome, were consistent with underlying differences in the abundance of distinctive sets of functional categories. These conclusions were robust with respect to plant configuration, operational and environmental conditions, and even differences in laboratory protocols. IMPORTANCE This work contributes to the elucidation of drivers of microbial community assembly in wastewater treatment systems. Our results are significant because they provide clear evidence that bacterial communities in WWTPs assemble mainly according to influent wastewater characteristics. Differences in bacterial community structures between WWTPs were consistent with differences in the abundance of distinctive sets of functional categories, which were related to the metabolic potential that would be expected according to the source of the wastewater. PMID:27316957

Blueberry virus A

USDA-ARS?s Scientific Manuscript database

Leaf yellowing on highbush blueberry ‘Spartan’ prompted Isogai et al. to investigate whether a virus was the causal agent of the disorder. After double-stranded RNA extraction from symptomatic material they identified a single band of 17 Kb, indicative of virus infection. Shotgun cloning and sequenc...

Shotgun proteomics of the barley seed proteome

USDA-ARS?s Scientific Manuscript database

Barley seed proteins are of prime importance to the brewing industry, human and animal nutrition and in plant breeding for cultivar identification. To obtain comprehensive proteomic data from barley seeds, acetone precipitated proteins were in-solution digested and the resulting peptides were analyz...

50 CFR 21.48 - Depredation order for double-crested cormorants to protect public resources.

Code of Federal Regulations, 2012 CFR

2012-10-01

... asphyxiation. Persons using shotguns must use nontoxic shot, as listed in 50 CFR 20.21(j). Persons using egg... control activities in a given year, provide a one-time written notice to the appropriate Service Regional...

50 CFR 21.48 - Depredation order for double-crested cormorants to protect public resources.

Code of Federal Regulations, 2014 CFR

2014-10-01

... asphyxiation. (i) Persons using shotguns must use nontoxic shot, as listed in 50 CFR 20.21(j). (ii) Beginning... activities in a given year, provide a one-time written notice to the appropriate Service Regional Migratory...

iProphet: Multi-level Integrative Analysis of Shotgun Proteomic Data Improves Peptide and Protein Identification Rates and Error Estimates*

PubMed Central

Shteynberg, David; Deutsch, Eric W.; Lam, Henry; Eng, Jimmy K.; Sun, Zhi; Tasman, Natalie; Mendoza, Luis; Moritz, Robert L.; Aebersold, Ruedi; Nesvizhskii, Alexey I.

2011-01-01

The combination of tandem mass spectrometry and sequence database searching is the method of choice for the identification of peptides and the mapping of proteomes. Over the last several years, the volume of data generated in proteomic studies has increased dramatically, which challenges the computational approaches previously developed for these data. Furthermore, a multitude of search engines have been developed that identify different, overlapping subsets of the sample peptides from a particular set of tandem mass spectrometry spectra. We present iProphet, the new addition to the widely used open-source suite of proteomic data analysis tools Trans-Proteomics Pipeline. Applied in tandem with PeptideProphet, it provides more accurate representation of the multilevel nature of shotgun proteomic data. iProphet combines the evidence from multiple identifications of the same peptide sequences across different spectra, experiments, precursor ion charge states, and modified states. It also allows accurate and effective integration of the results from multiple database search engines applied to the same data. The use of iProphet in the Trans-Proteomics Pipeline increases the number of correctly identified peptides at a constant false discovery rate as compared with both PeptideProphet and another state-of-the-art tool Percolator. As the main outcome, iProphet permits the calculation of accurate posterior probabilities and false discovery rate estimates at the level of sequence identical peptide identifications, which in turn leads to more accurate probability estimates at the protein level. Fully integrated with the Trans-Proteomics Pipeline, it supports all commonly used MS instruments, search engines, and computer platforms. The performance of iProphet is demonstrated on two publicly available data sets: data from a human whole cell lysate proteome profiling experiment representative of typical proteomic data sets, and from a set of Streptococcus pyogenes experiments more representative of organism-specific composite data sets. PMID:21876204

Sequencing degraded DNA from non-destructively sampled museum specimens for RAD-tagging and low-coverage shotgun phylogenetics.

PubMed

Tin, Mandy Man-Ying; Economo, Evan Philip; Mikheyev, Alexander Sergeyevich

2014-01-01

Ancient and archival DNA samples are valuable resources for the study of diverse historical processes. In particular, museum specimens provide access to biotas distant in time and space, and can provide insights into ecological and evolutionary changes over time. However, archival specimens are difficult to handle; they are often fragile and irreplaceable, and typically contain only short segments of denatured DNA. Here we present a set of tools for processing such samples for state-of-the-art genetic analysis. First, we report a protocol for minimally destructive DNA extraction of insect museum specimens, which produced sequenceable DNA from all of the samples assayed. The 11 specimens analyzed had fragmented DNA, rarely exceeding 100 bp in length, and could not be amplified by conventional PCR targeting the mitochondrial cytochrome oxidase I gene. Our approach made these samples amenable to analysis with commonly used next-generation sequencing-based molecular analytic tools, including RAD-tagging and shotgun genome re-sequencing. First, we used museum ant specimens from three species, each with its own reference genome, for RAD-tag mapping. Were able to use the degraded DNA sequences, which were sequenced in full, to identify duplicate reads and filter them prior to base calling. Second, we re-sequenced six Hawaiian Drosophila species, with millions of years of divergence, but with only a single available reference genome. Despite a shallow coverage of 0.37 ± 0.42 per base, we could recover a sufficient number of overlapping SNPs to fully resolve the species tree, which was consistent with earlier karyotypic studies, and previous molecular studies, at least in the regions of the tree that these studies could resolve. Although developed for use with degraded DNA, all of these techniques are readily applicable to more recent tissue, and are suitable for liquid handling automation.

Impact of library preparation protocols and template quantity on the metagenomic reconstruction of a mock microbial community

DOE PAGES

Bowers, Robert M.; Clum, Alicia; Tice, Hope; ...

2015-10-24

Background: The rapid development of sequencing technologies has provided access to environments that were either once thought inhospitable to life altogether or that contain too few cells to be analyzed using genomics approaches. While 16S rRNA gene microbial community sequencing has revolutionized our understanding of community composi tion and diversity over time and space, it only provides a crude estimate of microbial functional and metabolic potential. Alternatively, shotgun metagenomics allows comprehensive sampling of all genetic material in an environment, without any underlying primer biases. Until recently, one of the major bottlenecks of shotgun metagenomics has been the requirement for largemore » initial DNA template quantities during library preparation. Results: Here, we investigate the effects of varying template concentrations across three low biomass library preparation protocols on their ability to accurately reconstruct a mock microbial community of known composition. We analyze the effects of input DNA quantity and library preparation method on library insert size, GC content, community composition, assembly quality and metagenomic binning. We found that library preparation method and the amount of starting material had significant impacts on the mock community metagenomes. In particular, GC content shifted towards more GC rich sequences at the lower input quantities regardless of library prep method, the number of low quality reads that could not be mapped to the reference genomes increased with decreasing input quantities, and the different library preparation methods had an impact on overall metagenomic community composition. Conclusions: This benchmark study provides recommendations for library creation of representative and minimally biased metagenome shotgun sequencing, enabling insights into functional attributes of low biomass ecosystem microbial communities.« less

High-resolution phylogenetic microbial community profiling

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

Singer, Esther; Coleman-Derr, Devin; Bowman, Brett

2014-03-17

The representation of bacterial and archaeal genome sequences is strongly biased towards cultivated organisms, which belong to merely four phylogenetic groups. Functional information and inter-phylum level relationships are still largely underexplored for candidate phyla, which are often referred to as microbial dark matter. Furthermore, a large portion of the 16S rRNA gene records in the GenBank database are labeled as environmental samples and unclassified, which is in part due to low read accuracy, potential chimeric sequences produced during PCR amplifications and the low resolution of short amplicons. In order to improve the phylogenetic classification of novel species and advance ourmore » knowledge of the ecosystem function of uncultivated microorganisms, high-throughput full length 16S rRNA gene sequencing methodologies with reduced biases are needed. We evaluated the performance of PacBio single-molecule real-time (SMRT) sequencing in high-resolution phylogenetic microbial community profiling. For this purpose, we compared PacBio and Illumina metagenomic shotgun and 16S rRNA gene sequencing of a mock community as well as of an environmental sample from Sakinaw Lake, British Columbia. Sakinaw Lake is known to contain a large age of microbial species from candidate phyla. Sequencing results show that community structure based on PacBio shotgun and 16S rRNA gene sequences is highly similar in both the mock and the environmental communities. Resolution power and community representation accuracy from SMRT sequencing data appeared to be independent of GC content of microbial genomes and was higher when compared to Illumina-based metagenome shotgun and 16S rRNA gene (iTag) sequences, e.g. full-length sequencing resolved all 23 OTUs in the mock community, while iTags did not resolve closely related species. SMRT sequencing hence offers various potential benefits when characterizing uncharted microbial communities.« less

Impact of library preparation protocols and template quantity on the metagenomic reconstruction of a mock microbial community

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

Bowers, Robert M.; Clum, Alicia; Tice, Hope

Background: The rapid development of sequencing technologies has provided access to environments that were either once thought inhospitable to life altogether or that contain too few cells to be analyzed using genomics approaches. While 16S rRNA gene microbial community sequencing has revolutionized our understanding of community composi tion and diversity over time and space, it only provides a crude estimate of microbial functional and metabolic potential. Alternatively, shotgun metagenomics allows comprehensive sampling of all genetic material in an environment, without any underlying primer biases. Until recently, one of the major bottlenecks of shotgun metagenomics has been the requirement for largemore » initial DNA template quantities during library preparation. Results: Here, we investigate the effects of varying template concentrations across three low biomass library preparation protocols on their ability to accurately reconstruct a mock microbial community of known composition. We analyze the effects of input DNA quantity and library preparation method on library insert size, GC content, community composition, assembly quality and metagenomic binning. We found that library preparation method and the amount of starting material had significant impacts on the mock community metagenomes. In particular, GC content shifted towards more GC rich sequences at the lower input quantities regardless of library prep method, the number of low quality reads that could not be mapped to the reference genomes increased with decreasing input quantities, and the different library preparation methods had an impact on overall metagenomic community composition. Conclusions: This benchmark study provides recommendations for library creation of representative and minimally biased metagenome shotgun sequencing, enabling insights into functional attributes of low biomass ecosystem microbial communities.« less

Palynostratigraphic correlation of the Fort Union formation (Paleocene) in the Wind River Reservation and Waltman Area, Wind River Basin, Wyoming

USGS Publications Warehouse

Nichols, D.J.; Flores, R.M.

1993-01-01

Palynologic samples of the Fort Union Formation (Paleocene) were collected from selected composite measured sections and a drill hole in the Wind River Basin. Intervals studied are in outcrop in the Wind River Reservation in the Shotgun Butte area and near Hudson, Wyoming, and in the subsurface in the Muddy Ridge field. Age determinations were made using the palynostratigraphic zonation originally developed near Waltman, Wyoming, in the northeastern part of the basin. Although parts of the zonation have been called into question by other recent work in the basin, only the basal Paleocene biozone (P1) is in need of modification, and the zonation remains an effective tool for age determination and correlation in these rocks. Age control is sparse in parts of some sections studied, mostly because of a dominance of sandstone and conglomerate facies, but palynostratigraphy permits correlation of the study areas with each other and with the Waltman reference section.Palynology supports the following correlations: The lower unnamed member of the Fort Union Formation in the Shotgun Butte area correlates stratigraphically and chronologically with the lower part of the formation in the subsurface at Muddy Ridge field, with a short interval in the Hudson area, and with the lower and middle parts of the unnamed member in the Waltman area. The Shotgun Member correlates chronologically with the coal-bearing upper part of the Fort Union Formation in the subsurface at Muddy Ridge field and with the upper part of the unnamed member and Waltman Shale Member in the Waltman area. These correlations show that significant coal deposits in the subsurface have little or no expression in outcrop because of changes in facies.

Lipidomic and proteomic analysis of exosomes from mouse cortical collecting duct cells.

PubMed

Dang, Viet D; Jella, Kishore Kumar; Ragheb, Ragy R T; Denslow, Nancy D; Alli, Abdel A

2017-12-01

Exosomes are endosome-derived nanovesicles that are involved in cellular communication and signaling. Exosomes are produced by epithelial cells and are found in biologic fluids including blood and urine. The packaged material within exosomes includes proteins and lipids, but the molecular comparison within exosome subtypes is largely unknown. The purpose of this study was to investigate differences between exosomes derived from the apical plasma membrane and basolateral plasma membrane of polarized murine cortical collecting duct principal cells. Nanoparticle tracking analysis showed that the size and concentration of apical and basolateral exosomes remained relatively stable across 3 different temperatures (23, 37, and 42°C). Liquid chromatography-tandem mass spectrometry analysis revealed marked differences between the proteins packaged within the two types of exosomes from the same cells. Several proteins expressed at the inner leaflet of the plasma membrane, including α-actinin-1, moesin, 14-3-3 protein ζ/δ, annexin A1/A3/A4/A5/A6, clathrin heavy chain 1, glyceraldehyde-3-phosphate dehydrogenase, α-enolase, filamin-A, and heat shock protein 90, were identified in samples of apical plasma membrane-derived exosomes, but not in basolateral plasma membrane exosomes from mouse cortical collecting duct cells. In addition to differences at the protein level, mass spectrometry-based shotgun lipidomics analysis showed significant differences in the lipid classes and fatty acid composition of the two types of exosomes. We found higher levels of sphingomyelin and lower levels of cardiolipin, among other phospholipids in the apical plasma membrane compared to the basolateral plasma membrane exosomes. The molecular analyses of exosome subtypes presented herein will contribute to our understanding of exosome biogenesis, and the results may have potential implications for biomarker discovery.-Dang, V. D., Jella, K. K., Ragheb, R. R. T., Denslow, N. D., Alli, A. A. Lipidomic and proteomic analysis of exosomes from mouse cortical collecting duct cells. © FASEB.

Shotgun Metagenomics and Volatilome Profile of the Microbiota of Fermented Sausages.

PubMed

Ferrocino, Ilario; Bellio, Alberto; Giordano, Manuela; Macori, Guerrino; Romano, Angelo; Rantsiou, Kalliopi; Decastelli, Lucia; Cocolin, Luca

2018-02-01

Changes in the microbial gene content and abundance can be analyzed to detect shifts in the microbiota composition due to the use of a starter culture in the food fermentation process, with the consequent shift of key metabolic pathways directly connected with product acceptance. Meat fermentation is a complex process involving microbes that metabolize the main components in meat. The breakdown of carbohydrates, proteins, and lipids can lead to the formation of volatile organic compounds (VOCs) that can drastically affect the organoleptic characteristics of the final products. The present meta-analysis, performed with the shotgun DNA metagenomic approach, focuses on studying the microbiota and its gene content in an Italian fermented sausage produced by using a commercial starter culture (a mix of Lactobacillus sakei and Staphylococcus xylosus ), with the aim to discover the connections between the microbiota, microbiome, and the release of volatile metabolites during ripening. The inoculated fermentation with the starter culture limited the development of Enterobacteriaceae and reduced the microbial diversity compared to that from spontaneous fermentation. KEGG database genes associated with the reduction of acetaldehyde to ethanol (EC 1.1.1.1), acetyl phosphate to acetate (EC 2.7.2.1), and 2,3-butanediol to acetoin (EC 1.1.1.4) were most abundant in inoculated samples (I) compared to those in spontaneous fermentation samples (S). The volatilome profiles were highly consistent with the abundance of the genes; elevated acetic acid (1,173.85 μg/kg), ethyl acetate (251.58 μg/kg), and acetoin (1,100.19 μg/kg) were observed in the presence of the starters at the end of fermentation. Significant differences were found in the liking of samples based on flavor and odor, suggesting a higher preference by consumers for the spontaneous fermentation samples. Inoculated samples exhibited the lowest scores for the liking data, which were clearly associated with the highest concentration of acetic acid. IMPORTANCE We present an advance in the understanding of meat fermentation by coupling DNA sequencing metagenomics and metabolomics approaches to describe the microbial function during this process. Very few studies using this global approach have been dedicated to food, and none have examined sausage fermentation, underlying the originality of the study. The starter culture drastically affected the organoleptic properties of the products. This finding underlines the importance of starter culture selection that takes into consideration the functional characteristics of the microorganism to optimize production efficiency and product quality. Copyright © 2018 American Society for Microbiology.

Shotgun Canceling.

ERIC Educational Resources Information Center

Szymanski, Theodore

1999-01-01

Discusses a common misunderstanding demonstrated by many students in basic mathematics courses: not knowing how to properly "cancel" factors in simplifying mathematical equations. Asserts that "crossing-out" or "canceling" is not a valid mathematical operation, and that instructors should be wary about using these terms because of the ease with…

Comparison between Procedures using Sodium Dodecyl Sulfate for Shotgun Proteomic Analyses of Complex Samples

PubMed Central

Bereman, Michael S.; Egertson, Jarrett D.; MacCoss, Michael J.

2012-01-01

Filter aided sample preparation (FASP) and a new sample preparation method using a modified commercial SDS removal spin column are quantitatively compared in terms of their performance for shotgun proteomic experiments in three complex proteomic samples: a Saccharomyces cerevisiae lysate (insoluble fraction), a Caenorhabditis elegans lysate (soluble fraction), and a human embryonic kidney cell line (HEK293T). The characteristics and total number of peptides and proteins identified are compared between the two procedures. The SDS spin column procedure affords a conservative 4-fold improvement in throughput, is more reproducible, less expensive (i.e., requires less materials), and identifies between 30–107% more peptides at a q≤0.01, than the FASP procedure. The peptides identified by SDS spin column are more hydrophobic than species identified by the FASP procedure as indicated by the distribution of GRAVY scores. Ultimately, these improvements correlate to as great as a 50% increase in protein identifications with 2 or more peptides. PMID:21656683

Mspire-Simulator: LC-MS shotgun proteomic simulator for creating realistic gold standard data.

PubMed

Noyce, Andrew B; Smith, Rob; Dalgleish, James; Taylor, Ryan M; Erb, K C; Okuda, Nozomu; Prince, John T

2013-12-06

The most important step in any quantitative proteomic pipeline is feature detection (aka peak picking). However, generating quality hand-annotated data sets to validate the algorithms, especially for lower abundance peaks, is nearly impossible. An alternative for creating gold standard data is to simulate it with features closely mimicking real data. We present Mspire-Simulator, a free, open-source shotgun proteomic simulator that goes beyond previous simulation attempts by generating LC-MS features with realistic m/z and intensity variance along with other noise components. It also includes machine-learned models for retention time and peak intensity prediction and a genetic algorithm to custom fit model parameters for experimental data sets. We show that these methods are applicable to data from three different mass spectrometers, including two fundamentally different types, and show visually and analytically that simulated peaks are nearly indistinguishable from actual data. Researchers can use simulated data to rigorously test quantitation software, and proteomic researchers may benefit from overlaying simulated data on actual data sets.

Efficient marginalization to compute protein posterior probabilities from shotgun mass spectrometry data

PubMed Central

Serang, Oliver; MacCoss, Michael J.; Noble, William Stafford

2010-01-01

The problem of identifying proteins from a shotgun proteomics experiment has not been definitively solved. Identifying the proteins in a sample requires ranking them, ideally with interpretable scores. In particular, “degenerate” peptides, which map to multiple proteins, have made such a ranking difficult to compute. The problem of computing posterior probabilities for the proteins, which can be interpreted as confidence in a protein’s presence, has been especially daunting. Previous approaches have either ignored the peptide degeneracy problem completely, addressed it by computing a heuristic set of proteins or heuristic posterior probabilities, or by estimating the posterior probabilities with sampling methods. We present a probabilistic model for protein identification in tandem mass spectrometry that recognizes peptide degeneracy. We then introduce graph-transforming algorithms that facilitate efficient computation of protein probabilities, even for large data sets. We evaluate our identification procedure on five different well-characterized data sets and demonstrate our ability to efficiently compute high-quality protein posteriors. PMID:20712337

Fine-scale variation in meiotic recombination in Mimulus inferred from population shotgun sequencing

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

Hellsten, Uffe; Wright, Kevin M.; Jenkins, Jerry

2013-11-13

Meiotic recombination rates can vary widely across genomes, with hotspots of intense activity interspersed among cold regions. In yeast, hotspots tend to occur in promoter regions of genes, whereas in humans and mice hotspots are largely defined by binding sites of the PRDM9 protein. To investigate the detailed recombination pattern in a flowering plant we use shotgun resequencing of a wild population of the monkeyflower Mimulus guttatus to precisely locate over 400,000 boundaries of historic crossovers or gene conversion tracts. Their distribution defines some 13,000 hotspots of varying strengths, interspersed with cold regions of undetectably low recombination. Average recombination ratesmore » peak near starts of genes and fall off sharply, exhibiting polarity. Within genes, recombination tracts are more likely to terminate in exons than in introns. The general pattern is similar to that observed in yeast, as well as in PRDM9-knockout mice, suggesting that recombination initiation described here in Mimulus may reflect ancient and conserved eukaryotic mechanisms« less

High-throughput automated microfluidic sample preparation for accurate microbial genomics

PubMed Central

Kim, Soohong; De Jonghe, Joachim; Kulesa, Anthony B.; Feldman, David; Vatanen, Tommi; Bhattacharyya, Roby P.; Berdy, Brittany; Gomez, James; Nolan, Jill; Epstein, Slava; Blainey, Paul C.

2017-01-01

Low-cost shotgun DNA sequencing is transforming the microbial sciences. Sequencing instruments are so effective that sample preparation is now the key limiting factor. Here, we introduce a microfluidic sample preparation platform that integrates the key steps in cells to sequence library sample preparation for up to 96 samples and reduces DNA input requirements 100-fold while maintaining or improving data quality. The general-purpose microarchitecture we demonstrate supports workflows with arbitrary numbers of reaction and clean-up or capture steps. By reducing the sample quantity requirements, we enabled low-input (∼10,000 cells) whole-genome shotgun (WGS) sequencing of Mycobacterium tuberculosis and soil micro-colonies with superior results. We also leveraged the enhanced throughput to sequence ∼400 clinical Pseudomonas aeruginosa libraries and demonstrate excellent single-nucleotide polymorphism detection performance that explained phenotypically observed antibiotic resistance. Fully-integrated lab-on-chip sample preparation overcomes technical barriers to enable broader deployment of genomics across many basic research and translational applications. PMID:28128213

Plasticity in the proteome of Emiliania huxleyi CCMP 1516 to extremes of light is highly targeted.

PubMed

McKew, Boyd A; Lefebvre, Stephane C; Achterberg, Eric P; Metodieva, Gergana; Raines, Christine A; Metodiev, Metodi V; Geider, Richard J

2013-10-01

Optimality principles are often applied in theoretical studies of microalgal ecophysiology to predict changes in allocation of resources to different metabolic pathways, and optimal acclimation is likely to involve changes in the proteome, which typically accounts for > 50% of cellular nitrogen (N). We tested the hypothesis that acclimation of the microalga Emiliania huxleyi CCMP 1516 to suboptimal vs supraoptimal light involves large changes in the proteome as cells rebalance the capacities to absorb light, fix CO2 , perform biosynthesis and resist photooxidative stress. Emiliania huxleyi was grown in nutrient-replete continuous culture at 30 (LL) and 1000 μmol photons m(-2) s(-1) (HL), and changes in the proteome were assessed by LC-MS/MS shotgun proteomics. Changes were most evident in proteins involved in the light reactions of photosynthesis; the relative abundance of photosystem I (PSI) and PSII proteins was 70% greater in LL, light-harvesting fucoxanthin-chlorophyll proteins (Lhcfs) were up to 500% greater in LL and photoprotective LI818 proteins were 300% greater in HL. The marked changes in the abundances of Lhcfs and LI818s, together with the limited plasticity in the bulk of the E. huxleyi proteome, probably reflect evolutionary pressures to provide energy to maintain metabolic capabilities in stochastic light environments encountered by this species in nature. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Human gut microbiota community structures in urban and rural populations in Russia

PubMed Central

Tyakht, Alexander V.; Kostryukova, Elena S.; Popenko, Anna S.; Belenikin, Maxim S.; Pavlenko, Alexander V.; Larin, Andrey K.; Karpova, Irina Y.; Selezneva, Oksana V.; Semashko, Tatyana A.; Ospanova, Elena A.; Babenko, Vladislav V.; Maev, Igor V.; Cheremushkin, Sergey V.; Kucheryavyy, Yuriy A.; Shcherbakov, Petr L.; Grinevich, Vladimir B.; Efimov, Oleg I.; Sas, Evgenii I.; Abdulkhakov, Rustam A.; Abdulkhakov, Sayar R.; Lyalyukova, Elena A.; Livzan, Maria A.; Vlassov, Valentin V.; Sagdeev, Renad Z.; Tsukanov, Vladislav V.; Osipenko, Marina F.; Kozlova, Irina V.; Tkachev, Alexander V.; Sergienko, Valery I.; Alexeev, Dmitry G.; Govorun, Vadim M.

2013-01-01

The microbial community of the human gut has a crucial role in sustaining host homeostasis. High-throughput DNA sequencing has delineated the structural and functional configurations of gut metagenomes in world populations. The microbiota of the Russian population is of particular interest to researchers, because Russia encompasses a uniquely wide range of environmental conditions and ethnogeographical cohorts. Here we conduct a shotgun metagenomic analysis of gut microbiota samples from 96 healthy Russian adult subjects, which reveals novel microbial community structures. The communities from several rural regions display similarities within each region and are dominated by the bacterial taxa associated with the healthy gut. Functional analysis shows that the metabolic pathways exhibiting differential abundance in the novel types are primarily associated with the trade-off between the Bacteroidetes and Firmicutes phyla. The specific signatures of the Russian gut microbiota are likely linked to the host diet, cultural habits and socioeconomic status. PMID:24036685

Peptidomic analysis reveals proteolytic activity of kefir microorganisms on bovine milk proteins

PubMed Central

Dallas, David C.; Citerne, Florine; Tian, Tian; Silva, Vitor L. M.; Kalanetra, Karen M.; Frese, Steven A.; Robinson, Randall C.; Mills, David A.; Barile, Daniela

2015-01-01

Scope The microorganisms that make up kefir grains are well known for lactose fermentation, but the extent to which they hydrolyze and consume milk proteins remains poorly understood. Peptidomics technologies were used to examine the proteolytic activity of kefir grains on bovine milk proteins. Methods and results Gel electrophoresis revealed substantial digestion of milk proteins by kefir grains, with mass spectrometric analysis showing the release of 609 protein fragments and alteration of the abundance of >1,500 peptides that derived from 27 milk proteins. Kefir contained 25 peptides identified from the literature as having biological activity, including those with antihypertensive, antimicrobial, immunomodulatory, opioid and anti-oxidative functions. 16S rRNA and shotgun metagenomic sequencing identified the principle taxa in the culture as Lactobacillus species. Conclusion The model kefir sample contained thousands of protein fragments released in part by kefir microorganisms and in part by native milk proteases. PMID:26616950

An evaluation of the accuracy and speed of metagenome analysis tools

PubMed Central

Lindgreen, Stinus; Adair, Karen L.; Gardner, Paul P.

2016-01-01

Metagenome studies are becoming increasingly widespread, yielding important insights into microbial communities covering diverse environments from terrestrial and aquatic ecosystems to human skin and gut. With the advent of high-throughput sequencing platforms, the use of large scale shotgun sequencing approaches is now commonplace. However, a thorough independent benchmark comparing state-of-the-art metagenome analysis tools is lacking. Here, we present a benchmark where the most widely used tools are tested on complex, realistic data sets. Our results clearly show that the most widely used tools are not necessarily the most accurate, that the most accurate tool is not necessarily the most time consuming, and that there is a high degree of variability between available tools. These findings are important as the conclusions of any metagenomics study are affected by errors in the predicted community composition and functional capacity. Data sets and results are freely available from http://www.ucbioinformatics.org/metabenchmark.html PMID:26778510

P-MartCancer-Interactive Online Software to Enable Analysis of Shotgun Cancer Proteomic Datasets.

PubMed

Webb-Robertson, Bobbie-Jo M; Bramer, Lisa M; Jensen, Jeffrey L; Kobold, Markus A; Stratton, Kelly G; White, Amanda M; Rodland, Karin D

2017-11-01

P-MartCancer is an interactive web-based software environment that enables statistical analyses of peptide or protein data, quantitated from mass spectrometry-based global proteomics experiments, without requiring in-depth knowledge of statistical programming. P-MartCancer offers a series of statistical modules associated with quality assessment, peptide and protein statistics, protein quantification, and exploratory data analyses driven by the user via customized workflows and interactive visualization. Currently, P-MartCancer offers access and the capability to analyze multiple cancer proteomic datasets generated through the Clinical Proteomics Tumor Analysis Consortium at the peptide, gene, and protein levels. P-MartCancer is deployed as a web service (https://pmart.labworks.org/cptac.html), alternatively available via Docker Hub (https://hub.docker.com/r/pnnl/pmart-web/). Cancer Res; 77(21); e47-50. ©2017 AACR . ©2017 American Association for Cancer Research.

Peptidomic analysis reveals proteolytic activity of kefir microorganisms on bovine milk proteins.

PubMed

Dallas, David C; Citerne, Florine; Tian, Tian; Silva, Vitor L M; Kalanetra, Karen M; Frese, Steven A; Robinson, Randall C; Mills, David A; Barile, Daniela

2016-04-15

The microorganisms that make up kefir grains are well known for lactose fermentation, but the extent to which they hydrolyze and consume milk proteins remains poorly understood. Peptidomics technologies were used to examine the proteolytic activity of kefir grains on bovine milk proteins. Gel electrophoresis revealed substantial digestion of milk proteins by kefir grains, with mass spectrometric analysis showing the release of 609 protein fragments and alteration of the abundance of >1500 peptides that derived from 27 milk proteins. Kefir contained 25 peptides identified from the literature as having biological activity, including those with antihypertensive, antimicrobial, immunomodulatory, opioid and anti-oxidative functions. 16S rRNA and shotgun metagenomic sequencing identified the principle taxa in the culture as Lactobacillus species. The model kefir sample contained thousands of protein fragments released in part by kefir microorganisms and in part by native milk proteases. Copyright © 2015 Elsevier Ltd. All rights reserved.

PanFP: Pangenome-based functional profiles for microbial communities

DOE PAGES

Jun, Se -Ran; Hauser, Loren John; Schadt, Christopher Warren; ...

2015-09-26

For decades there has been increasing interest in understanding the relationships between microbial communities and ecosystem functions. Current DNA sequencing technologies allows for the exploration of microbial communities in two principle ways: targeted rRNA gene surveys and shotgun metagenomics. For large study designs, it is often still prohibitively expensive to sequence metagenomes at both the breadth and depth necessary to statistically capture the true functional diversity of a community. Although rRNA gene surveys provide no direct evidence of function, they do provide a reasonable estimation of microbial diversity, while being a very cost effective way to screen samples of interestmore » for later shotgun metagenomic analyses. However, there is a great deal of 16S rRNA gene survey data currently available from diverse environments, and thus a need for tools to infer functional composition of environmental samples based on 16S rRNA gene survey data. As a result, we present a computational method called pangenome based functional profiles (PanFP), which infers functional profiles of microbial communities from 16S rRNA gene survey data for Bacteria and Archaea. PanFP is based on pangenome reconstruction of a 16S rRNA gene operational taxonomic unit (OTU) from known genes and genomes pooled from the OTU s taxonomic lineage. From this lineage, we derive an OTU functional profile by weighting a pangenome s functional profile with the OTUs abundance observed in a given sample. We validated our method by comparing PanFP to the functional profiles obtained from the direct shotgun metagenomic measurement of 65 diverse communities via Spearman correlation coefficients. These correlations improved with increasing sequencing depth, within the range of 0.8 0.9 for the most deeply sequenced Human Microbiome Project mock community samples. PanFP is very similar in performance to another recently released tool, PICRUSt, for almost all of survey data analysed here. But, our method is unique in that any OTU building method can be used, as opposed to being limited to closed reference OTU picking strategies against specific reference sequence databases. In conclusion, we developed an automated computational method, which derives an inferred functional profile based on the 16S rRNA gene surveys of microbial communities. The inferred functional profile provides a cost effective way to study complex ecosystems through predicted comparative functional metagenomes and metadata analysis. All PanFP source code and additional documentation are freely available online at GitHub.« less

PanFP: pangenome-based functional profiles for microbial communities.

PubMed

Jun, Se-Ran; Robeson, Michael S; Hauser, Loren J; Schadt, Christopher W; Gorin, Andrey A

2015-09-26

For decades there has been increasing interest in understanding the relationships between microbial communities and ecosystem functions. Current DNA sequencing technologies allows for the exploration of microbial communities in two principle ways: targeted rRNA gene surveys and shotgun metagenomics. For large study designs, it is often still prohibitively expensive to sequence metagenomes at both the breadth and depth necessary to statistically capture the true functional diversity of a community. Although rRNA gene surveys provide no direct evidence of function, they do provide a reasonable estimation of microbial diversity, while being a very cost-effective way to screen samples of interest for later shotgun metagenomic analyses. However, there is a great deal of 16S rRNA gene survey data currently available from diverse environments, and thus a need for tools to infer functional composition of environmental samples based on 16S rRNA gene survey data. We present a computational method called pangenome-based functional profiles (PanFP), which infers functional profiles of microbial communities from 16S rRNA gene survey data for Bacteria and Archaea. PanFP is based on pangenome reconstruction of a 16S rRNA gene operational taxonomic unit (OTU) from known genes and genomes pooled from the OTU's taxonomic lineage. From this lineage, we derive an OTU functional profile by weighting a pangenome's functional profile with the OTUs abundance observed in a given sample. We validated our method by comparing PanFP to the functional profiles obtained from the direct shotgun metagenomic measurement of 65 diverse communities via Spearman correlation coefficients. These correlations improved with increasing sequencing depth, within the range of 0.8-0.9 for the most deeply sequenced Human Microbiome Project mock community samples. PanFP is very similar in performance to another recently released tool, PICRUSt, for almost all of survey data analysed here. But, our method is unique in that any OTU building method can be used, as opposed to being limited to closed-reference OTU picking strategies against specific reference sequence databases. We developed an automated computational method, which derives an inferred functional profile based on the 16S rRNA gene surveys of microbial communities. The inferred functional profile provides a cost effective way to study complex ecosystems through predicted comparative functional metagenomes and metadata analysis. All PanFP source code and additional documentation are freely available online at GitHub ( https://github.com/srjun/PanFP ).

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

Jun, Se -Ran; Hauser, Loren John; Schadt, Christopher Warren

For decades there has been increasing interest in understanding the relationships between microbial communities and ecosystem functions. Current DNA sequencing technologies allows for the exploration of microbial communities in two principle ways: targeted rRNA gene surveys and shotgun metagenomics. For large study designs, it is often still prohibitively expensive to sequence metagenomes at both the breadth and depth necessary to statistically capture the true functional diversity of a community. Although rRNA gene surveys provide no direct evidence of function, they do provide a reasonable estimation of microbial diversity, while being a very cost effective way to screen samples of interestmore » for later shotgun metagenomic analyses. However, there is a great deal of 16S rRNA gene survey data currently available from diverse environments, and thus a need for tools to infer functional composition of environmental samples based on 16S rRNA gene survey data. As a result, we present a computational method called pangenome based functional profiles (PanFP), which infers functional profiles of microbial communities from 16S rRNA gene survey data for Bacteria and Archaea. PanFP is based on pangenome reconstruction of a 16S rRNA gene operational taxonomic unit (OTU) from known genes and genomes pooled from the OTU s taxonomic lineage. From this lineage, we derive an OTU functional profile by weighting a pangenome s functional profile with the OTUs abundance observed in a given sample. We validated our method by comparing PanFP to the functional profiles obtained from the direct shotgun metagenomic measurement of 65 diverse communities via Spearman correlation coefficients. These correlations improved with increasing sequencing depth, within the range of 0.8 0.9 for the most deeply sequenced Human Microbiome Project mock community samples. PanFP is very similar in performance to another recently released tool, PICRUSt, for almost all of survey data analysed here. But, our method is unique in that any OTU building method can be used, as opposed to being limited to closed reference OTU picking strategies against specific reference sequence databases. In conclusion, we developed an automated computational method, which derives an inferred functional profile based on the 16S rRNA gene surveys of microbial communities. The inferred functional profile provides a cost effective way to study complex ecosystems through predicted comparative functional metagenomes and metadata analysis. All PanFP source code and additional documentation are freely available online at GitHub.« less

Shotgun metabolomic approach based on mass spectrometry for hepatic mitochondria of mice under arsenic exposure.

PubMed

García-Sevillano, M A; García-Barrera, T; Navarro, F; Montero-Lobato, Z; Gómez-Ariza, J L

2015-04-01

Mass spectrometry (MS)-based toxicometabolomics requires analytical approaches for obtaining unbiased metabolic profiles. The present work explores the general application of direct infusion MS using a high mass resolution analyzer (a hybrid systems triple quadrupole-time-of-flight) and a complementary gas chromatography-MS analysis to mitochondria extracts from mouse hepatic cells, emphasizing on mitochondria isolation from hepatic cells with a commercial kit, sample treatment after cell lysis, comprehensive metabolomic analysis and pattern recognition from metabolic profiles. Finally, the metabolomic platform was successfully checked on a case-study based on the exposure experiment of mice Mus musculus to inorganic arsenic during 12 days. Endogenous metabolites alterations were recognized by partial least squares-discriminant analysis. Subsequently, metabolites were identified by combining MS/MS analysis and metabolomics databases. This work reports for the first time the effects of As-exposure on hepatic mitochondria metabolic pathways based on MS, and reveals disturbances in Krebs cycle, β-oxidation pathway, amino acids degradation and perturbations in creatine levels. This non-target analysis provides extensive metabolic information from mitochondrial organelle, which could be applied to toxicology, pharmacology and clinical studies.

Lipid raft proteome reveals that oxidative phosphorylation system is associated with the plasma membrane.

PubMed

Kim, Bong-Woo; Lee, Chang Seok; Yi, Jae-Sung; Lee, Joo-Hyung; Lee, Joong-Won; Choo, Hyo-Jung; Jung, Soon-Young; Kim, Min-Sik; Lee, Sang-Won; Lee, Myung-Shik; Yoon, Gyesoon; Ko, Young-Gyu

2010-12-01

Although accumulating proteomic analyses have supported the fact that mitochondrial oxidative phosphorylation (OXPHOS) complexes are localized in lipid rafts, which mediate cell signaling, immune response and host-pathogen interactions, there has been no in-depth study of the physiological functions of lipid-raft OXPHOS complexes. Here, we show that many subunits of OXPHOS complexes were identified from the lipid rafts of human adipocytes, C2C12 myotubes, Jurkat cells and surface biotin-labeled Jurkat cells via shotgun proteomic analysis. We discuss the findings of OXPHOS complexes in lipid rafts, the role of the surface ATP synthase complex as a receptor for various ligands and extracellular superoxide generation by plasma membrane oxidative phosphorylation complexes.

Proteomic analysis of the enterocyte brush border

PubMed Central

McConnell, Russell E.; Benesh, Andrew E.; Mao, Suli; Tabb, David L.

2011-01-01

The brush border domain at the apex of intestinal epithelial cells is the primary site of nutrient absorption in the intestinal tract and the primary surface of interaction with microbes that reside in the lumen. Because the brush border is positioned at such a critical physiological interface, we set out to create a comprehensive list of the proteins that reside in this domain using shotgun mass spectrometry. The resulting proteome contains 646 proteins with diverse functions. In addition to the expected collection of nutrient processing and transport components, we also identified molecules expected to function in the regulation of actin dynamics, membrane bending, and extracellular adhesion. These results provide a foundation for future studies aimed at defining the molecular mechanisms underpinning brush border assembly and function. PMID:21330445

Shotgun label-free quantitative proteomics of developing peanut (Arachis hypogaea L.) seed

USDA-ARS?s Scientific Manuscript database

Legume seeds and peanuts, in particular, are an inexpensive source of plant proteins and edible oil. Owing to their importance in global food security, it is necessary to understand the genetic, biochemical, and physiological mechanisms controlling seed quality and nutritive attributes. A comprehens...

Five Stances That Have Got to Go

ERIC Educational Resources Information Center

Zeigler, Earle F.

1973-01-01

The five stances in physical education that have to go are as follows: a) the shotgun approach'' to professional preparation; b) the athletics uber alles approach''; c) the women are all right in their place approach''; d) the body of knowledge approach'' and the password is treadmill' approach.''

50 CFR 32.20 - Alabama.

Code of Federal Regulations, 2012 CFR

2012-10-01

...). 5. We allow shotguns with #4 shot or smaller, rifles firing .22 caliber rimfire ammunition, or... State-approved hunter education course. One adult may supervise no more than two youths. 7. We prohibit.... One adult may supervise no more than one youth. 8. We prohibit participation in organized drives. D...

50 CFR 32.20 - Alabama.

Code of Federal Regulations, 2011 CFR

2011-10-01

...). 5. We allow shotguns with #4 shot or smaller, rifles firing .22 caliber rimfire ammunition, or... State-approved hunter education course. One adult may supervise no more than two youths. 7. We prohibit.... One adult may supervise no more than one youth. 8. We prohibit participation in organized drives. D...

50 CFR 32.20 - Alabama.

Code of Federal Regulations, 2010 CFR

2010-10-01

...). 5. We allow shotguns with #4 shot or smaller, rifles firing .22 caliber rimfire ammunition, or... State-approved hunter education course. One adult may supervise no more than two youths. 7. We prohibit.... One adult may supervise no more than one youth. 8. We prohibit participation in organized drives. D...

50 CFR 32.20 - Alabama.

Code of Federal Regulations, 2013 CFR

2013-10-01

...). 5. We allow shotguns with #4 shot or smaller, rifles firing .22 caliber rimfire ammunition, or... State-approved hunter education course. One adult may supervise no more than two youths. 7. We prohibit.... One adult may supervise no more than one youth. 8. We prohibit participation in organized drives. D...

Characterization of adult transcriptomes from the omnivorous lady beetle Coleomegilla maculata fed pollen or insect Egg Diet

USDA-ARS?s Scientific Manuscript database

32 reference transcriptome sequences described herein are filed with the National Center for Biotechnology Information (NCBI), GenBank Bioproject PRJNA236444. Transcriptome Shotgun Assembly (TSA) will also be submitted when upload instructions are received from gb-admin....

Discovery, Validation and Characterization of 1039 Cattle Single Nucleotide Polymorphisms

USDA-ARS?s Scientific Manuscript database

We identified approximately 13000 putative single nucleotide polymorphisms (SNPs) by comparison of repeat-masked BAC-end sequences from the cattle RPCI-42 BAC library with whole-genome shotgun contigs of cattle genome assembly Btau 1.0. Genotyping of a subset of these SNPs was performed on a panel ...

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

Wilson, A.; Hayes, J.

Reported is an extensive investigation of the vandal resistance properties of common solar glazing materials. Many implements of destruction, including shotgun, splitting maul, rocks, pry bar, gasoline and logging chain, were used to determine how much abuse these glazing materials might withstand. In the hands of seasoned vandals, the glazing materials are found to be vulnerable to abuse.

Hidden in the Middle: Culture, Value and Reward in Bioinformatics

ERIC Educational Resources Information Center

Lewis, Jamie; Bartlett, Andrew; Atkinson, Paul

2016-01-01

Bioinformatics--the so-called shotgun marriage between biology and computer science--is an interdiscipline. Despite interdisciplinarity being seen as a virtue, for having the capacity to solve complex problems and foster innovation, it has the potential to place projects and people in anomalous categories. For example, valorised…

A whole-genome assembly of the domestic cow, Bos taurus

USDA-ARS?s Scientific Manuscript database

Background: The genome of the domestic cow, Bos taurus, was sequenced using a mixture of hierarchical and whole-genome shotgun sequencing methods. Results: We have assembled the 35 million sequence reads and applied a variety of assembly improvement techniques, creating an assembly of 2.86 billion b...

27 CFR 478.28 - Transportation of destructive devices and certain firearms.

Code of Federal Regulations, 2010 CFR

2010-04-01

... destructive devices and certain firearms. 478.28 Section 478.28 Alcohol, Tobacco Products, and Firearms BUREAU... destructive devices and certain firearms. (a) The Director may authorize a person to transport in interstate or foreign commerce any destructive device, machine gun, short-barreled shotgun, or short-barreled...

Characterization of reniform nematode genome through shotgun sequencing

USDA-ARS?s Scientific Manuscript database

The reniform nematode (RN), a major agricultural pest particularly on cotton in the United States(U.S.), is among the major plant parasitic nematodes for which limited genomic information exists. In this study, over 380 Mb of sequence data were generated from four pooled adult female RN and assembl...

50 CFR 92.20 - Methods and means.

Code of Federal Regulations, 2014 CFR

2014-10-01

... birds: (a) Swivel guns, shotguns larger than 10 gauge, punt guns, battery guns, machine guns, fish hooks, poisons, drugs, explosives, or stupefying substances; (b) Shooting from a sinkbox or any other type of low...) Hunting from any type of aircraft; (d) Taking waterfowl and other species using live birds as decoys...

50 CFR 92.20 - Methods and means.

Code of Federal Regulations, 2012 CFR

2012-10-01

... birds: (a) Swivel guns, shotguns larger than 10 gauge, punt guns, battery guns, machine guns, fish hooks, poisons, drugs, explosives, or stupefying substances; (b) Shooting from a sinkbox or any other type of low...) Hunting from any type of aircraft; (d) Taking waterfowl and other species using live birds as decoys...

50 CFR 92.20 - Methods and means.

Code of Federal Regulations, 2011 CFR

2011-10-01

... birds: (a) Swivel guns, shotguns larger than 10 gauge, punt guns, battery guns, machine guns, fish hooks, poisons, drugs, explosives, or stupefying substances; (b) Shooting from a sinkbox or any other type of low...) Hunting from any type of aircraft; (d) Taking waterfowl and other species using live birds as decoys...

50 CFR 92.20 - Methods and means.

Code of Federal Regulations, 2013 CFR

2013-10-01

... birds: (a) Swivel guns, shotguns larger than 10 gauge, punt guns, battery guns, machine guns, fish hooks, poisons, drugs, explosives, or stupefying substances; (b) Shooting from a sinkbox or any other type of low...) Hunting from any type of aircraft; (d) Taking waterfowl and other species using live birds as decoys...

32 CFR 552.100 - Definitions.

Code of Federal Regulations, 2011 CFR

2011-07-01

... designed or redesigned, made or remade, modified or remodified to automatically fire more than one shot by..., incendiary, blank, shotgun, black powder, and shot). Items shall only be considered as ammunition when loaded... smooth bore either a number of ball shot or a single projectile for each single pull of the trigger. (j...

32 CFR 552.100 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-07-01

... designed or redesigned, made or remade, modified or remodified to automatically fire more than one shot by..., incendiary, blank, shotgun, black powder, and shot). Items shall only be considered as ammunition when loaded... smooth bore either a number of ball shot or a single projectile for each single pull of the trigger. (j...

27 CFR 53.11 - Meaning of terms.

Code of Federal Regulations, 2011 CFR

2011-04-01

..., such as rifles, carbines, machine guns, shotguns, or fowling pieces, from which a shot, bullet, or... respect of which the violation occurs. Pistols. Small projectile firearms which have a short one-hand..., made, and intended to be aimed and fired from one hand. The term does not include gadget devices, guns...

27 CFR 53.11 - Meaning of terms.

Code of Federal Regulations, 2012 CFR

2012-04-01

..., such as rifles, carbines, machine guns, shotguns, or fowling pieces, from which a shot, bullet, or... respect of which the violation occurs. Pistols. Small projectile firearms which have a short one-hand..., made, and intended to be aimed and fired from one hand. The term does not include gadget devices, guns...

32 CFR 552.100 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-07-01

... designed or redesigned, made or remade, modified or remodified to automatically fire more than one shot by..., incendiary, blank, shotgun, black powder, and shot). Items shall only be considered as ammunition when loaded... smooth bore either a number of ball shot or a single projectile for each single pull of the trigger. (j...

32 CFR 552.100 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-07-01

... designed or redesigned, made or remade, modified or remodified to automatically fire more than one shot by..., incendiary, blank, shotgun, black powder, and shot). Items shall only be considered as ammunition when loaded... smooth bore either a number of ball shot or a single projectile for each single pull of the trigger. (j...

27 CFR 53.11 - Meaning of terms.

Code of Federal Regulations, 2013 CFR

2013-04-01

..., such as rifles, carbines, machine guns, shotguns, or fowling pieces, from which a shot, bullet, or... respect of which the violation occurs. Pistols. Small projectile firearms which have a short one-hand..., made, and intended to be aimed and fired from one hand. The term does not include gadget devices, guns...

32 CFR 552.126 - Definitions.

Code of Federal Regulations, 2011 CFR

2011-07-01

... designed to shoot, or can be readily restored to shoot automatically more than one shot without manual..., trace, incendiary, blank, shotgun, black powder, and shot). Items shall only be considered as ammunition... one or more barrels when held in one hand, and having: (1) A chamber(s) as an integral part(s) of, or...

32 CFR 552.126 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-07-01

... designed to shoot, or can be readily restored to shoot automatically more than one shot without manual..., trace, incendiary, blank, shotgun, black powder, and shot). Items shall only be considered as ammunition... one or more barrels when held in one hand, and having: (1) A chamber(s) as an integral part(s) of, or...

50 CFR 32.62 - Tennessee.

Code of Federal Regulations, 2011 CFR

2011-10-01

... normal voice contact and under supervision of an adult age 21 or older, who possesses a license. One... shot when hunting with a shotgun (see § 32.2(k)). B. Upland Game Hunting. We allow hunting of squirrel... available at the refuge headquarters. 3. You may possess only approved nontoxic shot while hunting turkey...

32 CFR 552.126 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-07-01

... designed to shoot, or can be readily restored to shoot automatically more than one shot without manual..., trace, incendiary, blank, shotgun, black powder, and shot). Items shall only be considered as ammunition... one or more barrels when held in one hand, and having: (1) A chamber(s) as an integral part(s) of, or...

32 CFR 552.126 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-07-01

... designed to shoot, or can be readily restored to shoot automatically more than one shot without manual..., trace, incendiary, blank, shotgun, black powder, and shot). Items shall only be considered as ammunition... one or more barrels when held in one hand, and having: (1) A chamber(s) as an integral part(s) of, or...

27 CFR 53.11 - Meaning of terms.

Code of Federal Regulations, 2010 CFR

2010-04-01

..., such as rifles, carbines, machine guns, shotguns, or fowling pieces, from which a shot, bullet, or... respect of which the violation occurs. Pistols. Small projectile firearms which have a short one-hand..., made, and intended to be aimed and fired from one hand. The term does not include gadget devices, guns...

27 CFR 53.11 - Meaning of terms.

Code of Federal Regulations, 2014 CFR

2014-04-01

..., such as rifles, carbines, machine guns, shotguns, or fowling pieces, from which a shot, bullet, or... respect of which the violation occurs. Pistols. Small projectile firearms which have a short one-hand..., made, and intended to be aimed and fired from one hand. The term does not include gadget devices, guns...

Strain-Level Metagenomic Analysis of the Fermented Dairy Beverage Nunu Highlights Potential Food Safety Risks

PubMed Central

Walsh, Aaron M.; Crispie, Fiona; Daari, Kareem; O'Sullivan, Orla; Martin, Jennifer C.; Arthur, Cornelius T.; Claesson, Marcus J.; Scott, Karen P.

2017-01-01

ABSTRACT The rapid detection of pathogenic strains in food products is essential for the prevention of disease outbreaks. It has already been demonstrated that whole-metagenome shotgun sequencing can be used to detect pathogens in food but, until recently, strain-level detection of pathogens has relied on whole-metagenome assembly, which is a computationally demanding process. Here we demonstrated that three short-read-alignment-based methods, i.e., MetaMLST, PanPhlAn, and StrainPhlAn, could accurately and rapidly identify pathogenic strains in spinach metagenomes that had been intentionally spiked with Shiga toxin-producing Escherichia coli in a previous study. Subsequently, we employed the methods, in combination with other metagenomics approaches, to assess the safety of nunu, a traditional Ghanaian fermented milk product that is produced by the spontaneous fermentation of raw cow milk. We showed that nunu samples were frequently contaminated with bacteria associated with the bovine gut and, worryingly, we detected putatively pathogenic E. coli and Klebsiella pneumoniae strains in a subset of nunu samples. Ultimately, our work establishes that short-read-alignment-based bioinformatics approaches are suitable food safety tools, and we describe a real-life example of their utilization. IMPORTANCE Foodborne pathogens are responsible for millions of illnesses each year. Here we demonstrate that short-read-alignment-based bioinformatics tools can accurately and rapidly detect pathogenic strains in food products by using shotgun metagenomics data. The methods used here are considerably faster than both traditional culturing methods and alternative bioinformatics approaches that rely on metagenome assembly; therefore, they can potentially be used for more high-throughput food safety testing. Overall, our results suggest that whole-metagenome sequencing can be used as a practical food safety tool to prevent diseases or to link outbreaks to specific food products. PMID:28625983

Enrichment allows identification of diverse, rare elements in metagenomic resistome-virulome sequencing.

PubMed

Noyes, Noelle R; Weinroth, Maggie E; Parker, Jennifer K; Dean, Chris J; Lakin, Steven M; Raymond, Robert A; Rovira, Pablo; Doster, Enrique; Abdo, Zaid; Martin, Jennifer N; Jones, Kenneth L; Ruiz, Jaime; Boucher, Christina A; Belk, Keith E; Morley, Paul S

2017-10-17

Shotgun metagenomic sequencing is increasingly utilized as a tool to evaluate ecological-level dynamics of antimicrobial resistance and virulence, in conjunction with microbiome analysis. Interest in use of this method for environmental surveillance of antimicrobial resistance and pathogenic microorganisms is also increasing. In published metagenomic datasets, the total of all resistance- and virulence-related sequences accounts for < 1% of all sequenced DNA, leading to limitations in detection of low-abundance resistome-virulome elements. This study describes the extent and composition of the low-abundance portion of the resistome-virulome, using a bait-capture and enrichment system that incorporates unique molecular indices to count DNA molecules and correct for enrichment bias. The use of the bait-capture and enrichment system significantly increased on-target sequencing of the resistome-virulome, enabling detection of an additional 1441 gene accessions and revealing a low-abundance portion of the resistome-virulome that was more diverse and compositionally different than that detected by more traditional metagenomic assays. The low-abundance portion of the resistome-virulome also contained resistance genes with public health importance, such as extended-spectrum betalactamases, that were not detected using traditional shotgun metagenomic sequencing. In addition, the use of the bait-capture and enrichment system enabled identification of rare resistance gene haplotypes that were used to discriminate between sample origins. These results demonstrate that the rare resistome-virulome contains valuable and unique information that can be utilized for both surveillance and population genetic investigations of resistance. Access to the rare resistome-virulome using the bait-capture and enrichment system validated in this study can greatly advance our understanding of microbiome-resistome dynamics.

A framework for intelligent data acquisition and real-time database searching for shotgun proteomics.

PubMed

Graumann, Johannes; Scheltema, Richard A; Zhang, Yong; Cox, Jürgen; Mann, Matthias

2012-03-01

In the analysis of complex peptide mixtures by MS-based proteomics, many more peptides elute at any given time than can be identified and quantified by the mass spectrometer. This makes it desirable to optimally allocate peptide sequencing and narrow mass range quantification events. In computer science, intelligent agents are frequently used to make autonomous decisions in complex environments. Here we develop and describe a framework for intelligent data acquisition and real-time database searching and showcase selected examples. The intelligent agent is implemented in the MaxQuant computational proteomics environment, termed MaxQuant Real-Time. It analyzes data as it is acquired on the mass spectrometer, constructs isotope patterns and SILAC pair information as well as controls MS and tandem MS events based on real-time and prior MS data or external knowledge. Re-implementing a top10 method in the intelligent agent yields similar performance to the data dependent methods running on the mass spectrometer itself. We demonstrate the capabilities of MaxQuant Real-Time by creating a real-time search engine capable of identifying peptides "on-the-fly" within 30 ms, well within the time constraints of a shotgun fragmentation "topN" method. The agent can focus sequencing events onto peptides of specific interest, such as those originating from a specific gene ontology (GO) term, or peptides that are likely modified versions of already identified peptides. Finally, we demonstrate enhanced quantification of SILAC pairs whose ratios were poorly defined in survey spectra. MaxQuant Real-Time is flexible and can be applied to a large number of scenarios that would benefit from intelligent, directed data acquisition. Our framework should be especially useful for new instrument types, such as the quadrupole-Orbitrap, that are currently becoming available.

Heterosis-associated proteome analyses of maize (Zea mays L.) seminal roots by quantitative label-free LC-MS.

PubMed

Marcon, Caroline; Lamkemeyer, Tobias; Malik, Waqas Ahmed; Ungrue, Denise; Piepho, Hans-Peter; Hochholdinger, Frank

2013-11-20

Heterosis is the superior performance of heterozygous F1-hybrid plants compared to their homozygous genetically distinct parents. Seminal roots are embryonic roots that play an important role during early maize (Zea mays L.) seedling development. In the present study the most abundant soluble proteins of 2-4cm seminal roots of the reciprocal maize F1-hybrids B73×Mo17 and Mo17×B73 and their parental inbred lines B73 and Mo17 were quantified by label-free LC-MS/MS. In total, 1918 proteins were detected by this shot-gun approach. Among those, 970 were represented by at least two peptides and were further analyzed. Eighty-five proteins displayed non-additive accumulation in at least one hybrid. The functional category protein metabolism was the most abundant class of non-additive proteins represented by 27 proteins. Within this category 16 of 17 non-additively accumulated ribosomal proteins showed high or above high parent expression in seminal roots. These results imply that an increased protein synthesis rate in hybrids might be related to the early manifestation of hybrid vigor in seminal roots. In the present study a shot-gun proteomics approach allowed for the identification of 1917 proteins and analysis of 970 seminal root proteins of maize that were represented by at least 2 peptides. The comparison of proteome complexity of reciprocal hybrids and their parental inbred lines indicates an increased protein synthesis rate in hybrids that may contribute to the early manifestation of heterosis in seminal roots. This article is part of a Special Issue entitled: Translational Plant Proteomics. Copyright © 2013 Elsevier B.V. All rights reserved.

Detecting trace components in liquid chromatography/mass spectrometry data sets with two-dimensional wavelets

NASA Astrophysics Data System (ADS)

Compton, Duane C.; Snapp, Robert R.

2007-09-01

TWiGS (two-dimensional wavelet transform with generalized cross validation and soft thresholding) is a novel algorithm for denoising liquid chromatography-mass spectrometry (LC-MS) data for use in "shot-gun" proteomics. Proteomics, the study of all proteins in an organism, is an emerging field that has already proven successful for drug and disease discovery in humans. There are a number of constraints that limit the effectiveness of liquid chromatography-mass spectrometry (LC-MS) for shot-gun proteomics, where the chemical signals are typically weak, and data sets are computationally large. Most algorithms suffer greatly from a researcher driven bias, making the results irreproducible and unusable by other laboratories. We thus introduce a new algorithm, TWiGS, that removes electrical (additive white) and chemical noise from LC-MS data sets. TWiGS is developed to be a true two-dimensional algorithm, which operates in the time-frequency domain, and minimizes the amount of researcher bias. It is based on the traditional discrete wavelet transform (DWT), which allows for fast and reproducible analysis. The separable two-dimensional DWT decomposition is paired with generalized cross validation and soft thresholding. The Haar, Coiflet-6, Daubechie-4 and the number of decomposition levels are determined based on observed experimental results. Using a synthetic LC-MS data model, TWiGS accurately retains key characteristics of the peaks in both the time and m/z domain, and can detect peaks from noise of the same intensity. TWiGS is applied to angiotensin I and II samples run on a LC-ESI-TOF-MS (liquid-chromatography-electrospray-ionization) to demonstrate its utility for the detection of low-lying peaks obscured by noise.

Temporal Dynamics and Decay of Putatively Allochthonous and Autochthonous Viral Genotypes in Contrasting Freshwater Lakes

PubMed Central

Barbosa, Jorge G.; Brown, Julia M.; Donelan, Ryan P.; Eaglesham, James B.; Eggleston, Erin M.; LaBarre, Brenna A.

2012-01-01

Aquatic viruses play important roles in the biogeochemistry and ecology of lacustrine ecosystems; however, their composition, dynamics, and interactions with viruses of terrestrial origin are less extensively studied. We used a viral shotgun metagenomic approach to elucidate candidate autochthonous (i.e., produced within the lake) and allochthonous (i.e., washed in from other habitats) viral genotypes for a comparative study of their dynamics in lake waters. Based on shotgun metagenomes prepared from catchment soil and freshwater samples from two contrasting lakes (Cayuga Lake and Fayetteville Green Lake), we selected two putatively autochthonous viral genotypes (phycodnaviruses likely infecting algae and cyanomyoviruses likely infecting picocyanobacteria) and two putatively allochthonous viral genotypes (geminiviruses likely infecting terrestrial plants and circoviruses infecting unknown hosts but common in soil libraries) for analysis by genotype-specific quantitative PCR (TaqMan) applied to DNAs from viruses in the viral size fraction of lake plankton, i.e., 0.2 μm > virus > 0.02 μm. The abundance of autochthonous genotypes largely reflected expected host abundance, while the abundance of allochthonous genotypes corresponded with rainfall and storm events in the respective catchments, suggesting that viruses with these genotypes may have been transported to the lake in runoff. The decay rates of allochthonous and autochthonous genotypes, assessed in incubations where all potential hosts were killed, were generally lower (0.13 to 1.50% h−1) than those reported for marine virioplankton but similar to those for freshwater virioplankton. Both allochthonous and autochthonous viral genotypes were detected at higher concentrations in subsurface sediments than at the water-sediment interface. Our data indicate that putatively allochthonous viruses are present in lake plankton and sediments, where their temporal dynamics reflect active transport to the lake during hydrological events and then decay once there. PMID:22773646

Analytical Utility of Mass Spectral Binning in Proteomic Experiments by SPectral Immonium Ion Detection (SPIID)*

PubMed Central

Kelstrup, Christian D.; Frese, Christian; Heck, Albert J. R.; Olsen, Jesper V.; Nielsen, Michael L.

2014-01-01

Unambiguous identification of tandem mass spectra is a cornerstone in mass-spectrometry-based proteomics. As the study of post-translational modifications (PTMs) by means of shotgun proteomics progresses in depth and coverage, the ability to correctly identify PTM-bearing peptides is essential, increasing the demand for advanced data interpretation. Several PTMs are known to generate unique fragment ions during tandem mass spectrometry, the so-called diagnostic ions, which unequivocally identify a given mass spectrum as related to a specific PTM. Although such ions offer tremendous analytical advantages, algorithms to decipher MS/MS spectra for the presence of diagnostic ions in an unbiased manner are currently lacking. Here, we present a systematic spectral-pattern-based approach for the discovery of diagnostic ions and new fragmentation mechanisms in shotgun proteomics datasets. The developed software tool is designed to analyze large sets of high-resolution peptide fragmentation spectra independent of the fragmentation method, instrument type, or protease employed. To benchmark the software tool, we analyzed large higher-energy collisional activation dissociation datasets of samples containing phosphorylation, ubiquitylation, SUMOylation, formylation, and lysine acetylation. Using the developed software tool, we were able to identify known diagnostic ions by comparing histograms of modified and unmodified peptide spectra. Because the investigated tandem mass spectra data were acquired with high mass accuracy, unambiguous interpretation and determination of the chemical composition for the majority of detected fragment ions was feasible. Collectively we present a freely available software tool that allows for comprehensive and automatic analysis of analogous product ions in tandem mass spectra and systematic mapping of fragmentation mechanisms related to common amino acids. PMID:24895383

A Framework for Intelligent Data Acquisition and Real-Time Database Searching for Shotgun Proteomics*

PubMed Central

Graumann, Johannes; Scheltema, Richard A.; Zhang, Yong; Cox, Jürgen; Mann, Matthias

2012-01-01

In the analysis of complex peptide mixtures by MS-based proteomics, many more peptides elute at any given time than can be identified and quantified by the mass spectrometer. This makes it desirable to optimally allocate peptide sequencing and narrow mass range quantification events. In computer science, intelligent agents are frequently used to make autonomous decisions in complex environments. Here we develop and describe a framework for intelligent data acquisition and real-time database searching and showcase selected examples. The intelligent agent is implemented in the MaxQuant computational proteomics environment, termed MaxQuant Real-Time. It analyzes data as it is acquired on the mass spectrometer, constructs isotope patterns and SILAC pair information as well as controls MS and tandem MS events based on real-time and prior MS data or external knowledge. Re-implementing a top10 method in the intelligent agent yields similar performance to the data dependent methods running on the mass spectrometer itself. We demonstrate the capabilities of MaxQuant Real-Time by creating a real-time search engine capable of identifying peptides “on-the-fly” within 30 ms, well within the time constraints of a shotgun fragmentation “topN” method. The agent can focus sequencing events onto peptides of specific interest, such as those originating from a specific gene ontology (GO) term, or peptides that are likely modified versions of already identified peptides. Finally, we demonstrate enhanced quantification of SILAC pairs whose ratios were poorly defined in survey spectra. MaxQuant Real-Time is flexible and can be applied to a large number of scenarios that would benefit from intelligent, directed data acquisition. Our framework should be especially useful for new instrument types, such as the quadrupole-Orbitrap, that are currently becoming available. PMID:22171319

Prediction of the neuropeptidomes of members of the Astacidea (Crustacea, Decapoda) using publicly accessible transcriptome shotgun assembly (TSA) sequence data.

PubMed

Christie, Andrew E; Chi, Megan

2015-12-01

The decapod infraorder Astacidea is comprised of clawed lobsters and freshwater crayfish. Due to their economic importance and their use as models for investigating neurochemical signaling, much work has focused on elucidating their neurochemistry, particularly their peptidergic systems. Interestingly, no astacidean has been the subject of large-scale peptidomic analysis via in silico transcriptome mining, this despite growing transcriptomic resources for members of this taxon. Here, the publicly accessible astacidean transcriptome shotgun assembly data were mined for putative peptide-encoding transcripts; these sequences were used to predict the structures of mature neuropeptides. One hundred seventy-six distinct peptides were predicted for Procambarus clarkii, including isoforms of adipokinetic hormone-corazonin-like peptide (ACP), allatostatin A (AST-A), allatostatin B, allatostatin C (AST-C) bursicon α, bursicon β, CCHamide, crustacean hyperglycemic hormone (CHH)/ion transport peptide (ITP), diuretic hormone 31 (DH31), eclosion hormone (EH), FMRFamide-like peptide, GSEFLamide, intocin, leucokinin, neuroparsin, neuropeptide F, pigment dispersing hormone, pyrokinin, RYamide, short neuropeptide F (sNPF), SIFamide, sulfakinin and tachykinin-related peptide (TRP). Forty-six distinct peptides, including isoforms of AST-A, AST-C, bursicon α, CCHamide, CHH/ITP, DH31, EH, intocin, myosuppressin, neuroparsin, red pigment concentrating hormone, sNPF and TRP, were predicted for Pontastacus leptodactylus, with a bursicon β and a neuroparsin predicted for Cherax quadricarinatus. The identification of ACP is the first from a decapod, while the predictions of CCHamide, EH, GSEFLamide, intocin, neuroparsin and RYamide are firsts for the Astacidea. Collectively, these data greatly expand the catalog of known astacidean neuropeptides and provide a foundation for functional studies of peptidergic signaling in members of this decapod infraorder. Copyright © 2015 Elsevier Inc. All rights reserved.

Partial Shotgun Sequencing of the Boechera stricta Genome Reveals Extensive Microsynteny and Promoter Conservation with Arabidopsis1[W

PubMed Central

Windsor, Aaron J.; Schranz, M. Eric; Formanová, Nataša; Gebauer-Jung, Steffi; Bishop, John G.; Schnabelrauch, Domenica; Kroymann, Juergen; Mitchell-Olds, Thomas

2006-01-01

Comparative genomics provides insight into the evolutionary dynamics that shape discrete sequences as well as whole genomes. To advance comparative genomics within the Brassicaceae, we have end sequenced 23,136 medium-sized insert clones from Boechera stricta, a wild relative of Arabidopsis (Arabidopsis thaliana). A significant proportion of these sequences, 18,797, are nonredundant and display highly significant similarity (BLASTn e-value ≤ 10−30) to low copy number Arabidopsis genomic regions, including more than 9,000 annotated coding sequences. We have used this dataset to identify orthologous gene pairs in the two species and to perform a global comparison of DNA regions 5′ to annotated coding regions. On average, the 500 nucleotides upstream to coding sequences display 71.4% identity between the two species. In a similar analysis, 61.4% identity was observed between 5′ noncoding sequences of Brassica oleracea and Arabidopsis, indicating that regulatory regions are not as diverged among these lineages as previously anticipated. By mapping the B. stricta end sequences onto the Arabidopsis genome, we have identified nearly 2,000 conserved blocks of microsynteny (bracketing 26% of the Arabidopsis genome). A comparison of fully sequenced B. stricta inserts to their homologous Arabidopsis genomic regions indicates that indel polymorphisms >5 kb contribute substantially to the genome size difference observed between the two species. Further, we demonstrate that microsynteny inferred from end-sequence data can be applied to the rapid identification and cloning of genomic regions of interest from nonmodel species. These results suggest that among diploid relatives of Arabidopsis, small- to medium-scale shotgun sequencing approaches can provide rapid and cost-effective benefits to evolutionary and/or functional comparative genomic frameworks. PMID:16607030

Tip Design of Hemodialysis Catheters Influences Thrombotic Events and Replacement Rate.

PubMed

Petridis, C; Nitschke, M; Lehne, W; Smith, E; Goltz, J P; Lehnert, H; Meier, M

2017-02-01

Central venous tunnelled hemodialysis catheters (CVTC) are used for initial vascular access in patients with renal failure. Tip design of the CVTC may play an important role in catheter function and complication rates, influencing adequate hemodialysis treatment of these patients. This prospective, observational cohort study compared the function and complication rates of two CVTCs in patients with end stage renal disease (ESRD) within a follow-up period of 24 months. The study included patients with ESRD who received either a CVTC with a split tip (ST) or a shotgun tip (SG). All patients underwent dialysis within 24 h of intervention. Blood flow was documented initially (Qb 0 ) and was followed up after 6 (Qb 6 ), 12 (Qb 12 ), and 24 (Qb 24 ) months. Analysis of blood flow and complication rates within the follow-up period was performed by questionnaires. In total, 185 patients were included, of whom 93 received a ST CVTC and 92 a SG CVTC. Baseline parameters did not differ significantly between groups. CVTC blood flow was not significantly different between the two devices. Thrombolytic therapy with Alteplase was used significantly more often in the ST group (29%) than in the SG group (16%) (p < 0.05). The CVTC replacement rate was significantly higher in the ST group (19.3%) compared with the SG group (8.7%) (p < 0.05). The tip design of CVTC (split or shotgun) appears to be irrelevant for long-term blood flow during dialysis treatment. However, patients may benefit from SG catheters over ST catheters where replacement rates and thrombolytic treatment are concerned. Copyright © 2016 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.

Noteworthy Facts about a Methane-Producing Microbial Community Processing Acidic Effluent from Sugar Beet Molasses Fermentation.

PubMed

Chojnacka, Aleksandra; Szczęsny, Paweł; Błaszczyk, Mieczysław K; Zielenkiewicz, Urszula; Detman, Anna; Salamon, Agnieszka; Sikora, Anna

2015-01-01

Anaerobic digestion is a complex process involving hydrolysis, acidogenesis, acetogenesis and methanogenesis. The separation of the hydrogen-yielding (dark fermentation) and methane-yielding steps under controlled conditions permits the production of hydrogen and methane from biomass. The characterization of microbial communities developed in bioreactors is crucial for the understanding and optimization of fermentation processes. Previously we developed an effective system for hydrogen production based on long-term continuous microbial cultures grown on sugar beet molasses. Here, the acidic effluent from molasses fermentation was used as the substrate for methanogenesis in an upflow anaerobic sludge blanket bioreactor. This study focused on the molecular analysis of the methane-yielding community processing the non-gaseous products of molasses fermentation. The substrate for methanogenesis produces conditions that favor the hydrogenotrophic pathway of methane synthesis. Methane production results from syntrophic metabolism whose key process is hydrogen transfer between bacteria and methanogenic Archaea. High-throughput 454 pyrosequencing of total DNA isolated from the methanogenic microbial community and bioinformatic sequence analysis revealed that the domain Bacteria was dominated by Firmicutes (mainly Clostridia), Bacteroidetes, δ- and γ-Proteobacteria, Cloacimonetes and Spirochaetes. In the domain Archaea, the order Methanomicrobiales was predominant, with Methanoculleus as the most abundant genus. The second and third most abundant members of the Archaeal community were representatives of the Methanomassiliicoccales and the Methanosarcinales. Analysis of the methanogenic sludge by scanning electron microscopy with Energy Dispersive X-ray Spectroscopy and X-ray diffraction showed that it was composed of small highly heterogeneous mineral-rich granules. Mineral components of methanogenic granules probably modulate syntrophic metabolism and methanogenic pathways. A rough functional analysis from shotgun data of the metagenome demonstrated that our knowledge of methanogenesis is poor and/or the enzymes responsible for methane production are highly effective, since despite reasonably good sequencing coverage, the details of the functional potential of the microbial community appeared to be incomplete.

Noteworthy Facts about a Methane-Producing Microbial Community Processing Acidic Effluent from Sugar Beet Molasses Fermentation

PubMed Central

Chojnacka, Aleksandra; Szczęsny, Paweł; Błaszczyk, Mieczysław K.; Zielenkiewicz, Urszula; Detman, Anna; Salamon, Agnieszka; Sikora, Anna

2015-01-01

Anaerobic digestion is a complex process involving hydrolysis, acidogenesis, acetogenesis and methanogenesis. The separation of the hydrogen-yielding (dark fermentation) and methane-yielding steps under controlled conditions permits the production of hydrogen and methane from biomass. The characterization of microbial communities developed in bioreactors is crucial for the understanding and optimization of fermentation processes. Previously we developed an effective system for hydrogen production based on long-term continuous microbial cultures grown on sugar beet molasses. Here, the acidic effluent from molasses fermentation was used as the substrate for methanogenesis in an upflow anaerobic sludge blanket bioreactor. This study focused on the molecular analysis of the methane-yielding community processing the non-gaseous products of molasses fermentation. The substrate for methanogenesis produces conditions that favor the hydrogenotrophic pathway of methane synthesis. Methane production results from syntrophic metabolism whose key process is hydrogen transfer between bacteria and methanogenic Archaea. High-throughput 454 pyrosequencing of total DNA isolated from the methanogenic microbial community and bioinformatic sequence analysis revealed that the domain Bacteria was dominated by Firmicutes (mainly Clostridia), Bacteroidetes, δ- and γ-Proteobacteria, Cloacimonetes and Spirochaetes. In the domain Archaea, the order Methanomicrobiales was predominant, with Methanoculleus as the most abundant genus. The second and third most abundant members of the Archaeal community were representatives of the Methanomassiliicoccales and the Methanosarcinales. Analysis of the methanogenic sludge by scanning electron microscopy with Energy Dispersive X-ray Spectroscopy and X-ray diffraction showed that it was composed of small highly heterogeneous mineral-rich granules. Mineral components of methanogenic granules probably modulate syntrophic metabolism and methanogenic pathways. A rough functional analysis from shotgun data of the metagenome demonstrated that our knowledge of methanogenesis is poor and/or the enzymes responsible for methane production are highly effective, since despite reasonably good sequencing coverage, the details of the functional potential of the microbial community appeared to be incomplete. PMID:26000448

OCaPPI-Db: an oligonucleotide probe database for pathogen identification through hybridization capture.

PubMed

Gasc, Cyrielle; Constantin, Antony; Jaziri, Faouzi; Peyret, Pierre

2017-01-01

The detection and identification of bacterial pathogens involved in acts of bio- and agroterrorism are essential to avoid pathogen dispersal in the environment and propagation within the population. Conventional molecular methods, such as PCR amplification, DNA microarrays or shotgun sequencing, are subject to various limitations when assessing environmental samples, which can lead to inaccurate findings. We developed a hybridization capture strategy that uses a set of oligonucleotide probes to target and enrich biomarkers of interest in environmental samples. Here, we present Oligonucleotide Capture Probes for Pathogen Identification Database (OCaPPI-Db), an online capture probe database containing a set of 1,685 oligonucleotide probes allowing for the detection and identification of 30 biothreat agents up to the species level. This probe set can be used in its entirety as a comprehensive diagnostic tool or can be restricted to a set of probes targeting a specific pathogen or virulence factor according to the user's needs. : http://ocappidb.uca.works. © The Author(s) 2017. Published by Oxford University Press.

Protein profiling of epidermal bladder cells from the halophyte Mesembryanthemum crystallinum.

PubMed

Barkla, Bronwyn J; Vera-Estrella, Rosario; Pantoja, Omar

2012-09-01

Plant epidermal trichomes are as varied in morphology as they are in function. In the halophyte Mesembryanthemum crystallinum, specialized trichomes called epidermal bladder cells (EBC) line the surface of leaves and stems, and increase dramatically in size and volume upon plant salt-treatment. These cells have been proposed to have roles in plant defense and UV protection, but primarily in sodium sequestration and as water reservoirs. To gain further understanding into the roles of EBC, a cell-type-specific proteomics approach was taken in which precision single-cell sampling of cell sap from individual EBC was combined with shotgun peptide sequencing (LC-MS/MS). Identified proteins showed diverse biological functions and cellular locations, with a high representation of proteins involved in H(+)-transport, carbohydrate metabolism, and photosynthesis. The proteome of EBC provides insight into the roles of these cells in ion and water homeostasis and raises the possibility that they are photosynthetically active and functioning in Crassulacean acid metabolism. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ecto-Fc MS identifies ligand-receptor interactions through extracellular domain Fc fusion protein baits and shotgun proteomic analysis

PubMed Central

Savas, Jeffrey N.; De Wit, Joris; Comoletti, Davide; Zemla, Roland; Ghosh, Anirvan

2015-01-01

Ligand-receptor interactions represent essential biological triggers which regulate many diverse and important cellular processes. We have developed a discovery-based proteomic biochemical protocol which couples affinity purification with multidimensional liquid chromatographic tandem mass spectrometry (LCLC-MS/MS) and bioinformatic analysis. Compared to previous approaches, our analysis increases sensitivity, shortens analysis duration, and boosts comprehensiveness. In this protocol, receptor extracellular domains are fused with the Fc region of IgG to generate fusion proteins that are purified from transfected HEK293T cells. These “ecto-Fcs” are coupled to protein A beads and serve as baits for binding assays with prey proteins extracted from rodent brain. After capture, the affinity purified proteins are digested into peptides and comprehensively analyzed by LCLC-MS/MS with ion trap mass spectrometers. In four working days, this protocol can generate shortlists of candidate ligand-receptor protein-protein interactions. Our “Ecto-Fc MS” approach outperforms antibody-based approaches and provides a reproducible and robust framework to identify extracellular ligand – receptor interactions. PMID:25101821

Comprehensive proteome analysis of nasal lavage samples after controlled exposure to welding nanoparticles shows an induced acute phase and a nuclear receptor, LXR/RXR, activation that influence the status of the extracellular matrix.

PubMed

Ali, Neserin; Ljunggren, Stefan; Karlsson, Helen M; Wierzbicka, Aneta; Pagels, Joakim; Isaxon, Christina; Gudmundsson, Anders; Rissler, Jenny; Nielsen, Jörn; Lindh, Christian H; Kåredal, Monica

2018-01-01

Epidemiological studies have shown that many welders experience respiratory symptoms. During the welding process a large number of airborne nanosized particles are generated, which might be inhaled and deposited in the respiratory tract. Knowledge of the underlying mechanisms behind observed symptoms is still partly lacking, although inflammation is suggested to play a central role. The aim of this study was to investigate the effects of welding fume particle exposure on the proteome expression level in welders suffering from respiratory symptoms, and changes in protein mediators in nasal lavage samples were analyzed. Such mediators will be helpful to clarify the pathomechanisms behind welding fume particle-induced effects. In an exposure chamber, 11 welders with work-related symptoms in the lower airways during the last month were exposed to mild-steel welding fume particles (1 mg/m 3 ) and to filtered air, respectively, in a double-blind manner. Nasal lavage samples were collected before, immediately after, and the day after exposure. The proteins in the nasal lavage were analyzed with two different mass spectrometry approaches, label-free discovery shotgun LC-MS/MS and a targeted selected reaction monitoring LC-MS/MS analyzing 130 proteins and four in vivo peptide degradation products. The analysis revealed 30 significantly changed proteins that were associated with two main pathways; activation of acute phase response signaling and activation of LXR/RXR, which is a nuclear receptor family involved in lipid signaling. Connective tissue proteins and proteins controlling the degradation of such tissues, including two different matrix metalloprotease proteins, MMP8 and MMP9, were among the significantly changed enzymes and were identified as important key players in the pathways. Exposure to mild-steel welding fume particles causes measurable changes on the proteome level in nasal lavage matrix in exposed welders, although no clinical symptoms were manifested. The results suggested that the exposure causes an immediate effect on the proteome level involving acute phase proteins and mediators regulating lipid signaling. Proteases involved in maintaining the balance between the formation and degradation of extracellular matrix proteins are important key proteins in the induced effects.

Molecular profiling of the Phytophthora plurivora secretome: a step towards understanding the cross-talk between plant pathogenic oomycetes and their hosts.

PubMed

Severino, Valeria; Farina, Annarita; Fleischmann, Frank; Dalio, Ronaldo J D; Di Maro, Antimo; Scognamiglio, Monica; Fiorentino, Antonio; Parente, Augusto; Osswald, Wolfgang; Chambery, Angela

2014-01-01

The understanding of molecular mechanisms underlying host-pathogen interactions in plant diseases is of crucial importance to gain insights on different virulence strategies of pathogens and unravel their role in plant immunity. Among plant pathogens, Phytophthora species are eliciting a growing interest for their considerable economical and environmental impact. Plant infection by Phytophthora phytopathogens is a complex process coordinated by a plethora of extracellular signals secreted by both host plants and pathogens. The characterization of the repertoire of effectors secreted by oomycetes has become an active area of research for deciphering molecular mechanisms responsible for host plants colonization and infection. Putative secreted proteins by Phytophthora species have been catalogued by applying high-throughput genome-based strategies and bioinformatic approaches. However, a comprehensive analysis of the effective secretome profile of Phytophthora is still lacking. Here, we report the first large-scale profiling of P. plurivora secretome using a shotgun LC-MS/MS strategy. To gain insight on the molecular signals underlying the cross-talk between plant pathogenic oomycetes and their host plants, we also investigate the quantitative changes of secreted protein following interaction of P. plurivora with the root exudate of Fagus sylvatica which is highly susceptible to the root pathogen. We show that besides known effectors, the expression and/or secretion levels of cell-wall-degrading enzymes were altered following the interaction with the host plant root exudate. In addition, a characterization of the F. sylvatica root exudate was performed by NMR and amino acid analysis, allowing the identification of the main released low-molecular weight components, including organic acids and free amino acids. This study provides important insights for deciphering the extracellular network involved in the highly susceptible P. plurivora-F. sylvatica interaction.

Subcellular proteome profiles of different latex fractions revealed washed solutions from rubber particles contain crucial enzymes for natural rubber biosynthesis.

PubMed

Wang, Dan; Sun, Yong; Chang, Lili; Tong, Zheng; Xie, Quanliang; Jin, Xiang; Zhu, Liping; He, Peng; Li, Hongbin; Wang, Xuchu

2018-06-30

Rubber particle (RP) is a specific organelle for natural rubber biosynthesis (NRB) and storage in rubber tree Hevea brasiliensis. NRB is processed by RP membrane-localized proteins, which were traditionally purified by repeated washing. However, we noticed many proteins in the discarded washing solutions (WS) from RP. Here, we compared the proteome profiles of WS, C-serum (CS) and RP by 2-DE, and identified 233 abundant proteins from WS by mass spectrometry. Many spots on 2-DE gels were identified as different protein species. We further performed shotgun analysis of CS, WS and RP and identified 1837, 1799 and 1020 unique proteins, respectively. Together with 2-DE, we finally identified 1825 proteins from WS, 246 were WS-specific. These WS-specific proteins were annotated in Gene Ontology, indicating most abundant pathways are organic substance metabolic process, protein degradation, primary metabolic process, and energy metabolism. Protein-protein interaction analysis revealed these WS-specific proteins are mainly involved in ribosomal metabolism, proteasome system, vacuolar protein sorting and endocytosis. Label free and Western blotting revealed many WS-specific proteins and protein complexes are crucial for NRB initiation. These findings not only deepen our understanding of WS proteome, but also provide new evidences on the roles of RP membrane proteins in NRB. Natural rubber is stored in rubber particle from the rubber tree. Rubber particles were traditionally purified by repeated washing, but many proteins were identified from the washing solutions (WS). We obtained the first visualization proteome profiles with 1825 proteins from WS, including 246 WS-specific ones. These WS proteins contain almost all enzymes for polyisoprene initiation and may play important roles in rubber biosynthesis. Copyright © 2018 Elsevier B.V. All rights reserved.

Metagenomic Analysis of the Microbiota from the Crop of an Invasive Snail Reveals a Rich Reservoir of Novel Genes

PubMed Central

Cardoso, Alexander M.; Cavalcante, Janaína J. V.; Cantão, Maurício E.; Thompson, Claudia E.; Flatschart, Roberto B.; Glogauer, Arnaldo; Scapin, Sandra M. N.; Sade, Youssef B.; Beltrão, Paulo J. M. S. I.; Gerber, Alexandra L.; Martins, Orlando B.; Garcia, Eloi S.; de Souza, Wanderley; Vasconcelos, Ana Tereza R.

2012-01-01

The shortage of petroleum reserves and the increase in CO2 emissions have raised global concerns and highlighted the importance of adopting sustainable energy sources. Second-generation ethanol made from lignocellulosic materials is considered to be one of the most promising fuels for vehicles. The giant snail Achatina fulica is an agricultural pest whose biotechnological potential has been largely untested. Here, the composition of the microbial population within the crop of this invasive land snail, as well as key genes involved in various biochemical pathways, have been explored for the first time. In a high-throughput approach, 318 Mbp of 454-Titanium shotgun metagenomic sequencing data were obtained. The predominant bacterial phylum found was Proteobacteria, followed by Bacteroidetes and Firmicutes. Viruses, Fungi, and Archaea were present to lesser extents. The functional analysis reveals a variety of microbial genes that could assist the host in the degradation of recalcitrant lignocellulose, detoxification of xenobiotics, and synthesis of essential amino acids and vitamins, contributing to the adaptability and wide-ranging diet of this snail. More than 2,700 genes encoding glycoside hydrolase (GH) domains and carbohydrate-binding modules were detected. When we compared GH profiles, we found an abundance of sequences coding for oligosaccharide-degrading enzymes (36%), very similar to those from wallabies and giant pandas, as well as many novel cellulase and hemicellulase coding sequences, which points to this model as a remarkable potential source of enzymes for the biofuel industry. Furthermore, this work is a major step toward the understanding of the unique genetic profile of the land snail holobiont. PMID:23133637

Multi-omics Analysis of Serum Samples Demonstrates Reprogramming of Organ Functions Via Systemic Calcium Mobilization and Platelet Activation in Metastatic Melanoma*

PubMed Central

Muqaku, Besnik; Eisinger, Martin; Meier, Samuel M.; Tahir, Ammar; Pukrop, Tobias; Haferkamp, Sebastian; Slany, Astrid; Reichle, Albrecht

2017-01-01

Pathophysiologies of cancer-associated syndromes such as cachexia are poorly understood and no routine biomarkers have been established, yet. Using shotgun proteomics, known marker molecules including PMEL, CRP, SAA, and CSPG4 were found deregulated in patients with metastatic melanoma. Targeted analysis of 58 selected proteins with multiple reaction monitoring was applied for independent data verification. In three patients, two of which suffered from cachexia, a tissue damage signature was determined, consisting of nine proteins, PLTP, CD14, TIMP1, S10A8, S10A9, GP1BA, PTPRJ, CD44, and C4A, as well as increased levels of glycine and asparagine, and decreased levels of polyunsaturated phosphatidylcholine concentrations, as determined by targeted metabolomics. Remarkably, these molecules are known to be involved in key processes of cancer cachexia. Based on these results, we propose a model how metastatic melanoma may lead to reprogramming of organ functions via formation of platelet activating factors from long-chain polyunsaturated phosphatidylcholines under oxidative conditions and via systemic induction of intracellular calcium mobilization. Calcium mobilization in platelets was demonstrated to alter levels of several of these marker molecules. Additionally, platelets from melanoma patients proved to be in a rather exhausted state, and platelet-derived eicosanoids implicated in tumor growth were found massively increased in blood from three melanoma patients. Platelets were thus identified as important source of serum protein and lipid alterations in late stage melanoma patients. As a result, the proposed model describes the crosstalk between lipolysis of fat tissue and muscle wasting mediated by oxidative stress, resulting in the metabolic deregulations characteristic for cachexia. PMID:27879288

A metagenomic-based survey of microbial (de)halogenation potential in a German forest soil

PubMed Central

Weigold, Pascal; El-Hadidi, Mohamed; Ruecker, Alexander; Huson, Daniel H.; Scholten, Thomas; Jochmann, Maik; Kappler, Andreas; Behrens, Sebastian

2016-01-01

In soils halogens (fluorine, chlorine, bromine, iodine) are cycled through the transformation of inorganic halides into organohalogen compounds and vice versa. There is evidence that these reactions are microbially driven but the key enzymes and groups of microorganisms involved are largely unknown. Our aim was to uncover the diversity, abundance and distribution of genes encoding for halogenating and dehalogenating enzymes in a German forest soil by shotgun metagenomic sequencing. Metagenomic libraries of three soil horizons revealed the presence of genera known to be involved in halogenation and dehalogenation processes such as Bradyrhizobium or Pseudomonas. We detected a so far unknown diversity of genes encoding for (de)halogenating enzymes in the soil metagenome including specific and unspecific halogenases as well as metabolic and cometabolic dehalogenases. Genes for non-heme, no-metal chloroperoxidases and haloalkane dehalogenases were the most abundant halogenase and dehalogenase genes, respectively. The high diversity and abundance of (de)halogenating enzymes suggests a strong microbial contribution to natural halogen cycling. This was also confirmed in microcosm experiments in which we quantified the biotic formation of chloroform and bromoform. Knowledge on microorganisms and genes that catalyze (de)halogenation reactions is critical because they are highly relevant to industrial biotechnologies and bioremediation applications. PMID:27353292

Penetrating abdominal trauma.

PubMed

Henneman, P L

1989-08-01

The management of patients with penetrating abdominal trauma is outlined in Figure 1. Patients with hemodynamic instability, evisceration, significant gastrointestinal bleeding, peritoneal signs, gunshot wounds with peritoneal violation, and type 2 and 3 shotgun wounds should undergo emergency laparotomy. The initial ED management of these patients includes airway management, monitoring of cardiac rhythm and vital signs, history, physical examination, and placement of intravenous lines. Blood should be obtained for initial hematocrit, type and cross-matching, electrolytes, and an alcohol level or drug screen as needed. Initial resuscitation should utilize crystalloid fluid replacement. If more than 2 liters of crystalloid are needed to stabilize an adult (less in a child), blood should be given. Group O Rh-negative packed red blood cells should be immediately available for a patient in impending arrest or massive hemorrhage. Type-specific blood should be available within 15 minutes. A patient with penetrating thoracic and high abdominal trauma should receive a portable chest x-ray, and a hemo- or pneumothorax should be treated with tube thoracostomy. An unstable patient with clinical signs consistent with a pneumothorax, however, should receive a tube thoracostomy prior to obtaining roentgenographic confirmation. If time permits, a nasogastric tube and Foley catheter should be placed, and the urine evaluated for blood (these procedures can be performed in the operating room). If kidney involvement is suspected because of hematuria or penetrating trauma in the area of a kidney or ureter in a patient requiring surgery, a single-shot IVP should be performed either in the ED or the operating room. An ECG is important in patients with possible cardiac involvement and in patients over the age of 40 going to the operating room. Tetanus status should be updated, and appropriate antibiotics covering bowel flora should be given. Operative management should rarely be delayed by procedures in the ED. Only lifesaving procedures necessary to prevent further deterioration should temporarily delay sending a patient to a waiting surgical team. Stable patients can be further evaluated in the ED. Those with stab wounds to the abdomen, flank, and selected cases of back injuries should undergo LWE. Those with negative LWE can be discharged after appropriate wound care and patient education. Patients with equivocal or positive LWE should undergo DPL. Patients with tangential gunshot wounds and possible type 2 shotgun injuries can undergo DPL. Table 8 lists the recommended thresholds for DPL. Patients with positive DPL should undergo exploration.(ABSTRACT TRUNCATED AT 400 WORDS)

Response of Burkholderia cenocepacia H111 to Micro-Oxia

PubMed Central

Pessi, Gabriella; Braunwalder, Rubina; Grunau, Alexander; Omasits, Ulrich; Ahrens, Christian H.; Eberl, Leo

2013-01-01

B. cenocepacia is an opportunistic human pathogen that is particularly problematic for patients suffering from cystic fibrosis (CF). In the CF lung bacteria grow to high densities within the viscous mucus that is limited in oxygen. Pseudomonas aeruginosa, the dominant pathogen in CF patients, is known to grow and survive under oxygen-limited to anaerobic conditions by using micro-oxic respiration, denitrification and fermentative pathways. In contrast, inspection of the genome sequences of available B. cenocepacia strains suggested that B. cenocepacia is an obligate aerobic and non-fermenting bacterium. In accordance with the bioinformatics analysis we observed that B. cenocepacia H111 is able to grow with as little as 0.1% O2 but not under strictly anoxic conditions. Phenotypic analyses revealed that H111 produced larger amounts of biofilm, pellicle and proteases under micro-oxic conditions (0.5%–5% O2, i.e. conditions that mimic those encountered in CF lung infection), and was more resistant to several antibiotics. RNA-Seq and shotgun proteomics analyses of cultures of B. cenocepacia H111 grown under micro-oxic and aerobic conditions showed up-regulation of genes involved in the synthesis of the exopolysaccharide (EPS) cepacian as well as several proteases, two isocitrate lyases and other genes potentially important for life in micro-oxia. Data deposition: RNA-Seq raw data files are accessible through the GEO Series accession number GSE48585. MS data have been deposited in the ProteomeXchange database (PXD000270). PMID:24023794

Proteomics and Pathway Analyses of the Milk Fat Globule in Sheep Naturally Infected by Mycoplasma agalactiae Provide Indications of the In Vivo Response of the Mammary Epithelium to Bacterial Infection ▿ ‡

PubMed Central

Addis, Maria Filippa; Pisanu, Salvatore; Ghisaura, Stefania; Pagnozzi, Daniela; Marogna, Gavino; Tanca, Alessandro; Biosa, Grazia; Cacciotto, Carla; Alberti, Alberto; Pittau, Marco; Roggio, Tonina; Uzzau, Sergio

2011-01-01

Milk fat globules (MFGs) are vesicles released in milk as fat droplets surrounded by the endoplasmic reticulum and apical cell membranes. During formation and apocrine secretion by lactocytes, various amounts of cytoplasmic crescents remain trapped within the released vesicle, making MFGs a natural sampling mechanism of the lactating cell contents. With the aim of investigating the events occurring in the mammary epithelium during bacterial infection, the MFG proteome was characterized by two-dimensional difference gel electrophoresis (2-D DIGE), SDS-PAGE followed by shotgun liquid chromatography-tandem mass spectrometry (GeLC-MS/MS), label-free quantification by the normalized spectral abundance factor (NSAF) approach, Western blotting, and pathway analysis, using sheep naturally infected by Mycoplasma agalactiae. A number of protein classes were found to increase in MFGs upon infection, including proteins involved in inflammation and host defense, cortical cytoskeleton proteins, heat shock proteins, and proteins related to oxidative stress. Conversely, a strikingly lower abundance was observed for proteins devoted to MFG metabolism and secretion. To our knowledge, this is the first report describing proteomic changes occurring in MFGs during sheep infectious mastitis. The results presented here offer new insights into the in vivo response of mammary epithelial cells to bacterial infection and open the way to the discovery of protein biomarkers for monitoring clinical and subclinical mastitis. PMID:21690237

Response of Burkholderia cenocepacia H111 to micro-oxia.

PubMed

Pessi, Gabriella; Braunwalder, Rubina; Grunau, Alexander; Omasits, Ulrich; Ahrens, Christian H; Eberl, Leo

2013-01-01

B. cenocepacia is an opportunistic human pathogen that is particularly problematic for patients suffering from cystic fibrosis (CF). In the CF lung bacteria grow to high densities within the viscous mucus that is limited in oxygen. Pseudomonas aeruginosa, the dominant pathogen in CF patients, is known to grow and survive under oxygen-limited to anaerobic conditions by using micro-oxic respiration, denitrification and fermentative pathways. In contrast, inspection of the genome sequences of available B. cenocepacia strains suggested that B. cenocepacia is an obligate aerobic and non-fermenting bacterium. In accordance with the bioinformatics analysis we observed that B. cenocepacia H111 is able to grow with as little as 0.1% O2 but not under strictly anoxic conditions. Phenotypic analyses revealed that H111 produced larger amounts of biofilm, pellicle and proteases under micro-oxic conditions (0.5%-5% O2, i.e. conditions that mimic those encountered in CF lung infection), and was more resistant to several antibiotics. RNA-Seq and shotgun proteomics analyses of cultures of B. cenocepacia H111 grown under micro-oxic and aerobic conditions showed up-regulation of genes involved in the synthesis of the exopolysaccharide (EPS) cepacian as well as several proteases, two isocitrate lyases and other genes potentially important for life in micro-oxia. RNA-Seq raw data files are accessible through the GEO Series accession number GSE48585. MS data have been deposited in the ProteomeXchange database (PXD000270).

High throughput techniques to reveal the molecular physiology and evolution of digestion in spiders.

PubMed

Fuzita, Felipe J; Pinkse, Martijn W H; Patane, José S L; Verhaert, Peter D E M; Lopes, Adriana R

2016-09-07

Spiders are known for their predatory efficiency and for their high capacity of digesting relatively large prey. They do this by combining both extracorporeal and intracellular digestion. Whereas many high throughput ("-omics") techniques focus on biomolecules in spider venom, so far this approach has not yet been applied to investigate the protein composition of spider midgut diverticula (MD) and digestive fluid (DF). We here report on our investigations of both MD and DF of the spider Nephilingis (Nephilengys) cruentata through the use of next generation sequencing and shotgun proteomics. This shows that the DF is composed of a variety of hydrolases including peptidases, carbohydrases, lipases and nuclease, as well as of toxins and regulatory proteins. We detect 25 astacins in the DF. Phylogenetic analysis of the corresponding transcript(s) in Arachnida suggests that astacins have acquired an unprecedented role for extracorporeal digestion in Araneae, with different orthologs used by each family. The results of a comparative study of spiders in distinct physiological conditions allow us to propose some digestion mechanisms in this interesting animal taxon. All the high throughput data allowed the demonstration that DF is a secretion originating from the MD. We identified enzymes involved in the extracellular and intracellular phases of digestion. Besides that, data analyses show a large gene duplication event in Araneae digestive process evolution, mainly of astacin genes. We were also able to identify proteins expressed and translated in the digestive system, which until now had been exclusively associated to venom glands.

Using "Omics" and Integrated Multi-Omics Approaches to Guide Probiotic Selection to Mitigate Chytridiomycosis and Other Emerging Infectious Diseases.

PubMed

Rebollar, Eria A; Antwis, Rachael E; Becker, Matthew H; Belden, Lisa K; Bletz, Molly C; Brucker, Robert M; Harrison, Xavier A; Hughey, Myra C; Kueneman, Jordan G; Loudon, Andrew H; McKenzie, Valerie; Medina, Daniel; Minbiole, Kevin P C; Rollins-Smith, Louise A; Walke, Jenifer B; Weiss, Sophie; Woodhams, Douglas C; Harris, Reid N

2016-01-01

Emerging infectious diseases in wildlife are responsible for massive population declines. In amphibians, chytridiomycosis caused by Batrachochytrium dendrobatidis, Bd, has severely affected many amphibian populations and species around the world. One promising management strategy is probiotic bioaugmentation of antifungal bacteria on amphibian skin. In vivo experimental trials using bioaugmentation strategies have had mixed results, and therefore a more informed strategy is needed to select successful probiotic candidates. Metagenomic, transcriptomic, and metabolomic methods, colloquially called "omics," are approaches that can better inform probiotic selection and optimize selection protocols. The integration of multiple omic data using bioinformatic and statistical tools and in silico models that link bacterial community structure with bacterial defensive function can allow the identification of species involved in pathogen inhibition. We recommend using 16S rRNA gene amplicon sequencing and methods such as indicator species analysis, the Kolmogorov-Smirnov Measure, and co-occurrence networks to identify bacteria that are associated with pathogen resistance in field surveys and experimental trials. In addition to 16S amplicon sequencing, we recommend approaches that give insight into symbiont function such as shotgun metagenomics, metatranscriptomics, or metabolomics to maximize the probability of finding effective probiotic candidates, which can then be isolated in culture and tested in persistence and clinical trials. An effective mitigation strategy to ameliorate chytridiomycosis and other emerging infectious diseases is necessary; the advancement of omic methods and the integration of multiple omic data provide a promising avenue toward conservation of imperiled species.

A filtering method to generate high quality short reads using illumina paired-end technology.

PubMed

Eren, A Murat; Vineis, Joseph H; Morrison, Hilary G; Sogin, Mitchell L

2013-01-01

Consensus between independent reads improves the accuracy of genome and transcriptome analyses, however lack of consensus between very similar sequences in metagenomic studies can and often does represent natural variation of biological significance. The common use of machine-assigned quality scores on next generation platforms does not necessarily correlate with accuracy. Here, we describe using the overlap of paired-end, short sequence reads to identify error-prone reads in marker gene analyses and their contribution to spurious OTUs following clustering analysis using QIIME. Our approach can also reduce error in shotgun sequencing data generated from libraries with small, tightly constrained insert sizes. The open-source implementation of this algorithm in Python programming language with user instructions can be obtained from https://github.com/meren/illumina-utils.

Gleaning evolutionary insights from the genome sequence of a probiotic yeast Saccharomyces boulardii

PubMed Central

2013-01-01

Background The yeast Saccharomyces boulardii is used worldwide as a probiotic to alleviate the effects of several gastrointestinal diseases and control antibiotics-associated diarrhea. While many studies report the probiotic effects of S. boulardii, no genome information for this yeast is currently available in the public domain. Results We report the 11.4 Mbp draft genome of this probiotic yeast. The draft genome was obtained by assembling Roche 454 FLX + shotgun data into 194 contigs with an N50 of 251 Kbp. We compare our draft genome with all other Saccharomyces cerevisiae genomes. Conclusions Our analysis confirms the close similarity of S. boulardii to S. cerevisiae strains and provides a framework to understand the probiotic effects of this yeast, which exhibits unique physiological and metabolic properties. PMID:24148866

SNP-VISTA: An Interactive SNPs Visualization Tool

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

Shah, Nameeta; Teplitsky, Michael V.; Pennacchio, Len A.

2005-07-05

Recent advances in sequencing technologies promise better diagnostics for many diseases as well as better understanding of evolution of microbial populations. Single Nucleotide Polymorphisms(SNPs) are established genetic markers that aid in the identification of loci affecting quantitative traits and/or disease in a wide variety of eukaryotic species. With today's technological capabilities, it is possible to re-sequence a large set of appropriate candidate genes in individuals with a given disease and then screen for causative mutations.In addition, SNPs have been used extensively in efforts to study the evolution of microbial populations, and the recent application of random shotgun sequencing to environmentalmore » samples makes possible more extensive SNP analysis of co-occurring and co-evolving microbial populations. The program is available at http://genome.lbl.gov/vista/snpvista.« less

Usefulness and limitations of postmortem computed tomography in forensic analysis of gunshot injuries: Three case reports.

PubMed

Usui, Akihito; Kawasumi, Yusuke; Hosokai, Yoshiyuki; Kozakai, Masataka; Saito, Haruo; Funayama, Masato

2016-01-01

Gunshot injury has always been an important field of investigation in postmortem forensic radiology. The localization and retrieval of the bullet and of potentially important fragments are vital to these cases. Using postmortem multidetector-row computed tomography (MDCT) prior to forensic autopsy, we sought to illustrate the importance of this modality in the noninvasive characterization of gunshot wounds. We obtained and analyzed MDCT images in three cases of gunshot wounds (accidental close-range shotgun shooting, suicidal contact gunshot to the head and accidental long-range buckshot shooting). We discuss the value of postmortem MDCT findings in gunshot wound cases by comparing with forensic autopsy findings in Japan, a developing country with miserably low autopsy rate. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Human Microbiome Acquisition and Bioinformatic Challenges in Metagenomic Studies

PubMed Central

2018-01-01

The study of the human microbiome has become a very popular topic. Our microbial counterpart, in fact, appears to play an important role in human physiology and health maintenance. Accordingly, microbiome alterations have been reported in an increasing number of human diseases. Despite the huge amount of data produced to date, less is known on how a microbial dysbiosis effectively contributes to a specific pathology. To fill in this gap, other approaches for microbiome study, more comprehensive than 16S rRNA gene sequencing, i.e., shotgun metagenomics and metatranscriptomics, are becoming more widely used. Methods standardization and the development of specific pipelines for data analysis are required to contribute to and increase our understanding of the human microbiome relationship with health and disease status. PMID:29382070

Analysis of the Proteome of Hair-Cell Stereocilia by Mass Spectrometry

PubMed Central

Krey, Jocelyn F.; Wilmarth, Philip A.; David, Larry L.; Barr-Gillespie, Peter G.

2017-01-01

Characterization of proteins that mediate mechanotransduction by hair cells, the sensory cells of the inner ear, is hampered by the scarcity of these cells and their sensory organelle, the hair bundle. Mass spectrometry, with its high sensitivity and identification precision, is the ideal method for determining which proteins are present in bundles and what proteins they interact with. We describe here the isolation of mouse hair bundles, as well as preparation of bundle-protein samples for mass spectrometry. We also describe protocols for data-dependent (shotgun) and parallel-reaction-monitoring (targeted) mass spectrometry that allow us to identify and quantify proteins of the hair bundle. These sensitive methods are particularly useful for comparing proteomes of wild-type and mice with deafness mutations affecting hair-bundle proteins. (120 words; maximum 250) PMID:28109437

Gleaning evolutionary insights from the genome sequence of a probiotic yeast Saccharomyces boulardii.

PubMed

Khatri, Indu; Akhtar, Akil; Kaur, Kamaldeep; Tomar, Rajul; Prasad, Gandham Satyanarayana; Ramya, Thirumalai Nallan Chakravarthy; Subramanian, Srikrishna

2013-10-22

The yeast Saccharomyces boulardii is used worldwide as a probiotic to alleviate the effects of several gastrointestinal diseases and control antibiotics-associated diarrhea. While many studies report the probiotic effects of S. boulardii, no genome information for this yeast is currently available in the public domain. We report the 11.4 Mbp draft genome of this probiotic yeast. The draft genome was obtained by assembling Roche 454 FLX + shotgun data into 194 contigs with an N50 of 251 Kbp. We compare our draft genome with all other Saccharomyces cerevisiae genomes. Our analysis confirms the close similarity of S. boulardii to S. cerevisiae strains and provides a framework to understand the probiotic effects of this yeast, which exhibits unique physiological and metabolic properties.

Draft genome sequence of chickpea (Cicer arietinum) provides a resource for trait improvement.

PubMed

Varshney, Rajeev K; Song, Chi; Saxena, Rachit K; Azam, Sarwar; Yu, Sheng; Sharpe, Andrew G; Cannon, Steven; Baek, Jongmin; Rosen, Benjamin D; Tar'an, Bunyamin; Millan, Teresa; Zhang, Xudong; Ramsay, Larissa D; Iwata, Aiko; Wang, Ying; Nelson, William; Farmer, Andrew D; Gaur, Pooran M; Soderlund, Carol; Penmetsa, R Varma; Xu, Chunyan; Bharti, Arvind K; He, Weiming; Winter, Peter; Zhao, Shancen; Hane, James K; Carrasquilla-Garcia, Noelia; Condie, Janet A; Upadhyaya, Hari D; Luo, Ming-Cheng; Thudi, Mahendar; Gowda, C L L; Singh, Narendra P; Lichtenzveig, Judith; Gali, Krishna K; Rubio, Josefa; Nadarajan, N; Dolezel, Jaroslav; Bansal, Kailash C; Xu, Xun; Edwards, David; Zhang, Gengyun; Kahl, Guenter; Gil, Juan; Singh, Karam B; Datta, Swapan K; Jackson, Scott A; Wang, Jun; Cook, Douglas R

2013-03-01

Chickpea (Cicer arietinum) is the second most widely grown legume crop after soybean, accounting for a substantial proportion of human dietary nitrogen intake and playing a crucial role in food security in developing countries. We report the ∼738-Mb draft whole genome shotgun sequence of CDC Frontier, a kabuli chickpea variety, which contains an estimated 28,269 genes. Resequencing and analysis of 90 cultivated and wild genotypes from ten countries identifies targets of both breeding-associated genetic sweeps and breeding-associated balancing selection. Candidate genes for disease resistance and agronomic traits are highlighted, including traits that distinguish the two main market classes of cultivated chickpea--desi and kabuli. These data comprise a resource for chickpea improvement through molecular breeding and provide insights into both genome diversity and domestication.

Exploiting long read sequencing technologies to establish high quality highly contiguous pig reference genome assemblies

USDA-ARS?s Scientific Manuscript database

The current pig reference genome sequence (Sscrofa10.2) was established using Sanger sequencing and following the clone-by-clone hierarchical shotgun sequencing approach used in the public human genome project. However, as sequence coverage was low (4-6x) the resulting assembly was only of draft qua...

27 CFR 478.112 - Importation by a licensed importer.

Code of Federal Regulations, 2010 CFR

2010-04-01

...) The type (e.g., rifle, shotgun, pistol, revolver and, in the case of ammunition only, ball, wadcutter, shot, etc.); (C) The caliber, gauge, or size; (D) The model; (E) The barrel length, if a firearm or... statement describing such intended use; or (C) If an unserviceable firearm (other than a machine gun) being...

27 CFR 478.113 - Importation by other licensees.

Code of Federal Regulations, 2010 CFR

2010-04-01

...) The type (e.g., rifle, shotgun, pistol, revolver and, in the case of ammunition only, ball, wadcutter, shot, etc.); (C) The caliber, gauge, or size; (D) The model; (E) The barrel length, if a firearm or... machine gun) being imported as a curio or museum piece, a description of how it was rendered unserviceable...

Nunatchiagmi (Stories about Buckland).

ERIC Educational Resources Information Center

Thomas, Evans

Printed in both Inupiat and English, this 32 page booklet recounts stories of native life in Buckland, Alaska. It is printed in large type and simply written; illustrations accompany each short narrative. Several stories are told by Evans Thomas who remembers his boyhood days as he fired a shotgun for the first time, shot his first seal, broke a…

27 CFR 479.93 - Transfers of firearms to certain persons.

Code of Federal Regulations, 2012 CFR

2012-04-01

..., FIREARMS, AND EXPLOSIVES, DEPARTMENT OF JUSTICE FIREARMS AND AMMUNITION MACHINE GUNS, DESTRUCTIVE DEVICES... persons. Where the transfer of a destructive device, machine gun, short-barreled shotgun, or short-barreled rifle is to be made by a person licensed under the provisions of Title I of the Gun Control Act of...

27 CFR 479.93 - Transfers of firearms to certain persons.

Code of Federal Regulations, 2011 CFR

2011-04-01

..., FIREARMS, AND EXPLOSIVES, DEPARTMENT OF JUSTICE FIREARMS AND AMMUNITION MACHINE GUNS, DESTRUCTIVE DEVICES... persons. Where the transfer of a destructive device, machine gun, short-barreled shotgun, or short-barreled rifle is to be made by a person licensed under the provisions of Title I of the Gun Control Act of...

27 CFR 479.93 - Transfers of firearms to certain persons.

Code of Federal Regulations, 2014 CFR

2014-04-01

..., FIREARMS, AND EXPLOSIVES, DEPARTMENT OF JUSTICE FIREARMS AND AMMUNITION MACHINE GUNS, DESTRUCTIVE DEVICES... persons. Where the transfer of a destructive device, machine gun, short-barreled shotgun, or short-barreled rifle is to be made by a person licensed under the provisions of Title I of the Gun Control Act of...

27 CFR 479.93 - Transfers of firearms to certain persons.

Code of Federal Regulations, 2010 CFR

2010-04-01

..., FIREARMS, AND EXPLOSIVES, DEPARTMENT OF JUSTICE FIREARMS AND AMMUNITION MACHINE GUNS, DESTRUCTIVE DEVICES... persons. Where the transfer of a destructive device, machine gun, short-barreled shotgun, or short-barreled rifle is to be made by a person licensed under the provisions of Title I of the Gun Control Act of...

27 CFR 479.93 - Transfers of firearms to certain persons.

Code of Federal Regulations, 2013 CFR

2013-04-01

..., FIREARMS, AND EXPLOSIVES, DEPARTMENT OF JUSTICE FIREARMS AND AMMUNITION MACHINE GUNS, DESTRUCTIVE DEVICES... persons. Where the transfer of a destructive device, machine gun, short-barreled shotgun, or short-barreled rifle is to be made by a person licensed under the provisions of Title I of the Gun Control Act of...

The American cranberry mitochondrial genome reveals the presence of selenocysteine (tRNA-Sec and SECIS) insertion machinery in land plants

USDA-ARS?s Scientific Manuscript database

The American cranberry (Vaccinium macrocarpon Ait.) mitochondrial genome was assembled and reconstructed from whole genome 454 Roche GS-FLX and Illumina shotgun sequences. Compared with other Asterids, the reconstruction of the genome revealed an average size mitochondrion (459,678 nt) with comparat...

A new rainbow trout (Oncorhynchus mykiss) reference genome assembly

USDA-ARS?s Scientific Manuscript database

In an effort to improve the rainbow trout reference genome assembly, we have re-sequenced the doubled-haploid Swanson line using the longest available reads from the Illumina technology. Overall we generated over 510 million 260nt paired-end shotgun reads, and 1 billion 160nt mate-pair reads from f...

Rhipicephalus (Boophilus) microplus strain Deutsch, whole genome shotgun sequencing project first submission of genome sequence

USDA-ARS?s Scientific Manuscript database

The size and repetitive nature of the Rhipicephalus microplus genome makes obtaining a full genome sequence difficult. Cot filtration/selection techniques were used to reduce the repetitive fraction of the tick genome and enrich for the fraction of DNA with gene-containing regions. The Cot-selected ...

Rhipicephalus microplus strain Deutsch, whole genome shotgun sequencing project Version 2

USDA-ARS?s Scientific Manuscript database

The cattle tick, Rhipicephalus (Boophilus) microplus, has a genome over 2.4 times the size of the human genome, and with over 70% of repetitive DNA, this genome would prove very costly to sequence at today's prices and difficult to assemble and analyze. Cot filtration/selection techniques were used ...

Sequencing of 15,622 gene-bearing BACs clarifies the gene-dense regions of the barley genome

USDA-ARS?s Scientific Manuscript database

Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole-genome shotgun sequences with a physical and genetic framework....

50 CFR 32.62 - Tennessee.

Code of Federal Regulations, 2012 CFR

2012-10-01

... older, who possesses a license. One adult hunter may supervise no more than two youth hunters. 9. You may possess only approved nontoxic shot while hunting with a shotgun (see § 32.2(k)). 10. We prohibit... nontoxic shot while hunting turkey (see § 32.2(k)). 4. Hunters may possess lead-rifled slugs while deer...

50 CFR 32.67 - Washington.

Code of Federal Regulations, 2010 CFR

2010-10-01

... nontoxic shot while in the field (see § 32.2(k)). 2. You may not shoot or discharge any firearm from... regulations subject to the following conditions: 1. You may only possess approved nontoxic shot while on the... possess only approved nontoxic shot while on the refuge. 2. We allow only shotguns and archery hunting. 3...

50 CFR 32.22 - Arizona.

Code of Federal Regulations, 2011 CFR

2011-10-01

... to a maximum three-shell capacity, unless it is plugged with a one-piece filler, incapable of removal... chapter). 2. You may possess only nontoxic shot while hunting in the field (see § 32.2(k)). 3. We prohibit... the following conditions: 1. We allow only shotguns. 2. You may possess only approved nontoxic shot...

50 CFR 32.43 - Mississippi.

Code of Federal Regulations, 2011 CFR

2011-10-01

... reverse side of the form and deposit it at one of the refuge information stations. Include all game... for details. 3. When hunting, we allow only shotguns with approved nontoxic shot (see § 32.2(k)), .17... card and deposit it at one of the hunter information stations. Include all game harvested, and if there...

50 CFR 32.22 - Arizona.

Code of Federal Regulations, 2012 CFR

2012-10-01

... to a maximum three-shell capacity, unless it is plugged with a one-piece filler, incapable of removal... chapter). 2. You may possess only nontoxic shot while hunting in the field (see § 32.2(k)). 3. We prohibit... the following conditions: 1. We allow only shotguns. 2. You may possess only approved nontoxic shot...

50 CFR 32.29 - Georgia.

Code of Federal Regulations, 2012 CFR

2012-10-01

... sunset of the second hunt day. 3. Each hunter may place one stand on the refuge no earlier than 1 month... license. One adult may supervise no more than one youth hunter. 18. We prohibit mooring boats to the.... We allow only nontoxic shot with the use of a shotgun in designated areas at the manager's discretion...

50 CFR 32.38 - Maine.

Code of Federal Regulations, 2010 CFR

2010-10-01

... articles of hunter-orange clothing or material. One article must be a solid-colored-hunter-orange hat; the... nontoxic shot when hunting with a shotgun (see § 32.2(k)). 3. We open the refuge to hunting during the... hunter must wear two articles of hunter-orange clothing or material. One article must be a solid-colored...

50 CFR 32.44 - Missouri.

Code of Federal Regulations, 2013 CFR

2013-10-01

... approved nontoxic shot (see § 32.2(k)). 2. We allow upland game hunting on the 131-acre mainland unit of... Statewide seasons using archery methods or shotguns using shot no larger than BB. C. Big Game Hunting. We... with shot no larger than BB. 6. You may possess only approved nontoxic shot while hunting on the refuge...

50 CFR 32.22 - Arizona.

Code of Federal Regulations, 2013 CFR

2013-10-01

... to a maximum three-shell capacity, unless it is plugged with a one-piece filler, incapable of removal... chapter). 2. You may possess only nontoxic shot while hunting in the field (see § 32.2(k)). 3. We prohibit... the following conditions: 1. We allow only shotguns. 2. You may possess only approved nontoxic shot...

50 CFR 32.43 - Mississippi.

Code of Federal Regulations, 2014 CFR

2014-10-01

... must complete the reverse side of the card/form and deposit it at one of the refuge information stations. Include all game harvested; if you harvested no game, report “0”. Hunters may possess only one.... 3. When hunting, we allow only shotguns with approved nontoxic shot (see § 32.2(k)), .17 or .22...

50 CFR 32.44 - Missouri.

Code of Federal Regulations, 2010 CFR

2010-10-01

... approved nontoxic shot (see § 32.2(k)). C. Big Game Hunting. We allow hunting of deer and turkey on... spring turkey hunting. We allow only shotguns with approved nontoxic shot (see § 32.2(k)). 7. We prohibit..., address, and phone number. 9. We allow only one tree stand per deer hunter. D. Sport Fishing. We allow...

50 CFR 32.55 - Oklahoma.

Code of Federal Regulations, 2010 CFR

2010-10-01

... hunt duck during designated refuge seasons. 6. You may only possess approved nontoxic shot while in the... during designated refuge seasons. 4. Shotgun hunters may only possess approved nontoxic shot while in the... shot while in the field (see § 32.2(k)). 2. We require hunters to check-in and out of the refuge. 3...

50 CFR 32.62 - Tennessee.

Code of Federal Regulations, 2014 CFR

2014-10-01

... older, who possesses a license. One adult hunter may supervise no more than two youth hunters. 9. You may possess only approved nontoxic shot while hunting with a shotgun (see § 32.2(k)). 10. We prohibit... nontoxic shot while hunting turkey (see § 32.2(k)). 4. Hunters may possess lead-rifled slugs while deer...

50 CFR 32.64 - Utah.

Code of Federal Regulations, 2010 CFR

2010-10-01

... approved nontoxic shot while in the field. 3. Hunters may not use pits or permanent blinds. 4. Airboats are... possess only approved nontoxic shot while in the field. 2. Pheasants may not be taken with a shotgun of any description capable of holding more than three shells, unless it is plugged with a one-piece...