Beyond the Thumbrule Approach: Regulatory Innovations for Bioprospecting in India

Directory of Open Access Journals (Sweden)

Kabir Sanjay Bavikatte and Morten Walløe Tvedt

2015-06-01

Full Text Available In the wake of the Nagoya Protocol on Access and Benefit Sharing (ABS, there is unprecedented attention on good Green Governance, which implies the development of resource efficient, rights based and effective domestic frameworks to regulate bioprospecting. India has been pioneering in this regard due to its ABS legislation in 2002 that long preceded the Nagoya Protocol. However much has changed since 2002 and while India has learnt a great deal from its ‘learning by doing’ method, there are valuable lessons that can be learnt from innovations in the ABS frameworks of other countries. The innovations of these countries are a great resource for National Biodiversity Authority (NBA in India that is seeking to make the processing of the bioprospecting applications in India more optimal. The current paper highlights the challenges faced by the NBA in processing the high volumes of bioprospecting applications, analyses the reasons for such challenges and proposes solutions for the same.

MEGGASENSE - The Metagenome/Genome Annotated Sequence Natural Language Search Engine: A Platform for 
the Construction of Sequence Data Warehouses.

Science.gov (United States)

Gacesa, Ranko; Zucko, Jurica; Petursdottir, Solveig K; Gudmundsdottir, Elisabet Eik; Fridjonsson, Olafur H; Diminic, Janko; Long, Paul F; Cullum, John; Hranueli, Daslav; Hreggvidsson, Gudmundur O; Starcevic, Antonio

2017-06-01

The MEGGASENSE platform constructs relational databases of DNA or protein sequences. The default functional analysis uses 14 106 hidden Markov model (HMM) profiles based on sequences in the KEGG database. The Solr search engine allows sophisticated queries and a BLAST search function is also incorporated. These standard capabilities were used to generate the SCATT database from the predicted proteome of Streptomyces cattleya . The implementation of a specialised metagenome database (AMYLOMICS) for bioprospecting of carbohydrate-modifying enzymes is described. In addition to standard assembly of reads, a novel 'functional' assembly was developed, in which screening of reads with the HMM profiles occurs before the assembly. The AMYLOMICS database incorporates additional HMM profiles for carbohydrate-modifying enzymes and it is illustrated how the combination of HMM and BLAST analyses helps identify interesting genes. A variety of different proteome and metagenome databases have been generated by MEGGASENSE.

Bioprospecting and evolving alternative xylose and arabinose pathway enzymes for use in Saccharomyces cerevisiae.

Science.gov (United States)

Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S

2016-03-01

Bioprospecting is an effective way to find novel enzymes from strains with desirable phenotypes. Such bioprospecting has enabled organisms such as Saccharomyces cerevisiae to utilize nonnative pentose sugars. Yet, the efficiency of this pentose catabolism (especially for the case of arabinose) remains suboptimal. Thus, further pathway optimization or identification of novel, optimal pathways is needed. Previously, we identified a novel set of xylan catabolic pathway enzymes from a superior pentose-utilizing strain of Ustilago bevomyces. These enzymes were used to successfully engineer a xylan-utilizing S. cerevisiae through a blended approach of bioprospecting and evolutionary engineering. Here, we expanded this approach to xylose and arabinose catabolic pathway engineering and demonstrated that bioprospected xylose and arabinose catabolic pathways from U. bevomyces offer alternative choices for enabling efficient pentose catabolism in S. cerevisiae. By introducing a novel set of xylose catabolic genes from U. bevomyces, growth rates were improved up to 85 % over a set of traditional Scheffersomyces stipitis pathway genes. In addition, we suggested an alternative arabinose catabolic pathway which, after directed evolution and pathway engineering, enabled S. cerevisiae to grow on arabinose as a sole carbon source in minimal medium with growth rates upwards of 0.05 h(-1). This pathway represents the most efficient growth of yeast on pure arabinose minimal medium. These pathways provide great starting points for further strain development and demonstrate the utility of bioprospecting from U. bevomyces.

Science Academies' Refresher Course on Bioprospection of ...

Indian Academy of Sciences (India)

2017-11-22

Nov 22, 2017 ... A refresher course on 'Bioprospection of Bioresources: Land to Lab Approach' will be held at PG and Research Department of Botany, St. Joseph's College, Tiruchirappalli, Tamil Nadu for two weeks from 04–18 January,2018. The aim of the Refresher Course is to encourage the College teachers and ...

Bioprospecting of Red Sea Sponges for Novel Antiviral Pharmacophores

KAUST Repository

O'Rourke, Aubrie

2015-01-01

the coast of Saudi Arabia serves as a newly accessible location, which provides the opportunity to bioprospect marine sponges with the purpose of identifying novel antiviral scaffolds. Antivirals are underrepresented in present day clinical trials, as well

Bioprospecting thermophiles for cellulase production: a review

OpenAIRE

Acharya,Somen; Chaudhary,Anita

2012-01-01

Most of the potential bioprospecting is currently related to the study of the extremophiles and their potential use in industrial processes. Recently microbial cellulases find applications in various industries and constitute a major group of industrial enzymes. Considerable amount of work has been done on microbial cellulases, especially with resurgence of interest in biomass ethanol production employing cellulases and use of cellulases in textile and paper industry. Most efficient method of...

Bioprospecting for bioactives from seaweeds: potential, obstacles and alternatives

Directory of Open Access Journals (Sweden)

Renato C. Pereira

2012-08-01

Full Text Available Seaweeds are potential sources of high biotechnological interest due to production of a great diversity of compounds exhibiting a broad spectrum of biological activities. On the other hand, there is an urgent need for management options for a sustainable approach to the use of marine organisms as a source of bioactive compounds. This review discusses the bioprospection for bioactive seaweed compounds as pharmaceuticals and antifouling agents, encompassing their potential and possible obstacles and alternatives. In spite of their potential, research on pharmaceuticals and antifouling agents from seaweeds includes mainly the search for molecules that exhibit these biological activities, but lacks of consideration of fundamental and limiting aspects such as the development of alternatives to sustainable supply. However, for the complete development of pharmaceuticals and antifouling compounds in Brazil, marine bioprospection should be more comprehensive, associating the search for molecules with an analysis of their supply. In this way, it is possible to promote sustainable development and conservation of biodiversity, as well as to assert the economic development of Brazil.

Bioprospecting for bioactives from seaweeds: potential, obstacles and alternatives

Directory of Open Access Journals (Sweden)

Renato C. Pereira

2012-06-01

Full Text Available Seaweeds are potential sources of high biotechnological interest due to production of a great diversity of compounds exhibiting a broad spectrum of biological activities. On the other hand, there is an urgent need for management options for a sustainable approach to the use of marine organisms as a source of bioactive compounds. This review discusses the bioprospection for bioactive seaweed compounds as pharmaceuticals and antifouling agents, encompassing their potential and possible obstacles and alternatives. In spite of their potential, research on pharmaceuticals and antifouling agents from seaweeds includes mainly the search for molecules that exhibit these biological activities, but lacks of consideration of fundamental and limiting aspects such as the development of alternatives to sustainable supply. However, for the complete development of pharmaceuticals and antifouling compounds in Brazil, marine bioprospection should be more comprehensive, associating the search for molecules with an analysis of their supply. In this way, it is possible to promote sustainable development and conservation of biodiversity, as well as to assert the economic development of Brazil.

Exploration of soil metagenome diversity for prospection of enzymes involved in lignocellulosic biomass conversion

Energy Technology Data Exchange (ETDEWEB)

Alvarez, T.M.; Squina, F.M. [Laboratorio Nacional de Luz Sincrotron (LNLS), Campinas, SP (Brazil); Paixao, D.A.A.; Franco Cairo, J.P.L.; Buchli, F.; Ruller, R. [Laboratorio Nacional de Ciencia e Tecnologia do Bioetanol (CTBE), Campinas, SP (Brazil); Prade, R. [Oklahoma State University, Sillwater, OK (United States)

2012-07-01

Full text: Metagenomics allows access to genetic information encoded in DNA of microorganisms recalcitrant to cultivation. They represent a reservoir of novel biocatalyst with potential application in environmental friendly techniques aiming to overcome the dependence on fossil fuels and also to diminish air and water pollution. The focus of our work is the generation of a tool kit of lignocellulolytic enzymes from soil metagenome, which could be used for second generation ethanol production. Environmental samples were collected at a sugarcane field after harvesting, where it is expected that the microbial population involved on lignocellulose degradation was enriched due to the presence of straws covering the soil. Sugarcane Bagasse-Degrading-Soil (SBDS) metagenome was massively-parallel-454-Roche-sequenced. We identified a full repertoire of genes with significant match to glycosyl hydrolases catalytic domain and carbohydrate-binding modules. Soil metagenomics libraries cloned into pUC19 were screened through functional assays. CMC-agar screening resulted in positive clones, revealing new cellulases coding genes. Through a CMC-zymogram it was possible to observe that one of these genes, nominated as E-1, corresponds to an enzyme that is secreted to the extracellular medium, suggesting that the cloned gene carried the original signal peptide. Enzymatic assays and analysis through capillary electrophoresis showed that E-1 was able to cleave internal glycosidic bonds of cellulose. New rounds of functional screenings through chromogenic substrates are being conducted aiming the generation of a library of lignocellulolytic enzymes derived from soil metagenome, which may become key component for development of second generation biofuels. (author)

Ten years of bioprospecting activities at the CSIR: BP4 as a model

CSIR Research Space (South Africa)

Fouché, Gerda

2008-11-01

Full Text Available The bioprospecting group of CSIR Biosciences, focuses on the transformation of African traditional medicines into minimally-processed, scientifically-validated herbal medicines and remedies; the discovery of new pharmaceutical active ingredients...

Glycosylation Engineering

DEFF Research Database (Denmark)

Clausen, Henrik; Wandall, Hans H.; Steentoft, Catharina

2017-01-01

Knowledge of the cellular pathways of glycosylation across phylogeny provides opportunities for designing glycans via genetic engineering in a wide variety of cell types including bacteria, fungi, plant cells, and mammalian cells. The commercial demand for glycosylation engineering is broad......, including production of biological therapeutics with defined glycosylation (Chapter 57). This chapter describes how knowledge of glycan structures and their metabolism (Parts I–III of this book) has led to the current state of glycosylation engineering in different cell types. Perspectives for rapid...

Bioprospecting Sediments from Red Sea Coastal Lagoons for Microorganisms and Their Antimicrobial Potential

KAUST Repository

Al Amoudi, Soha

2016-01-01

location with an increased probability of successful antibiotic bioprospecting, while the best sediment type in RHL for this purpose is microbial mat. Moreover, the phylum Actinobacteria tends to be the common target for research when it comes to antibiotic

Characterization of three plant biomass-degrading microbial consortia by metagenomics- and metasecretomics-based approaches

DEFF Research Database (Denmark)

Jiménez, Diego Javier; Brossi, Maria Julia de Lima; Schückel, Julia

2016-01-01

). The highest degradation rates of lignin (~59 %) were observed with SG-M, whereas CS-M showed a high consumption of cellulose and hemicellulose. Analyses of the carbohydrate-active enzymes in the three microbial consortia showed the dominance of glycosyl hydrolases (e.g. of families GH3, GH43, GH13, GH10, GH29......), switchgrass (SG-M) and corn stover (CS-M) under aerobic and mesophilic conditions. Molecular fingerprintings, bacterial 16S ribosomal RNA (rRNA) gene amplicon sequencing and metagenomic analyses showed that the three microbial consortia were taxonomically distinct. Based on the taxonomic affiliation...

Molecular cloning, expression, and characterization of four novel thermo-alkaliphilic enzymes retrieved from a metagenomic library.

Science.gov (United States)

Maruthamuthu, Mukil; van Elsas, Jan Dirk

2017-01-01

Enzyme discovery is a promising approach to aid in the deconstruction of recalcitrant plant biomass in an industrial process. Novel enzymes can be readily discovered by applying metagenomics on whole microbiomes. Our goal was to select, examine, and characterize eight novel glycoside hydrolases that were previously detected in metagenomic libraries, to serve biotechnological applications with high performance. Here, eight glycosyl hydrolase family candidate genes were selected from metagenomes of wheat straw-degrading microbial consortia using molecular cloning and subsequent gene expression studies in Escherichia coli. Four of the eight enzymes had significant activities on either p NP-β-d-galactopyranoside, p NP-β-d-xylopyranoside, p NP-α-l-arabinopyranoside or p NP-α-d-glucopyranoside. These proteins, denoted as proteins 1, 2, 5 and 6, were his-tag purified and their nature and activities further characterized using molecular and activity screens with the p NP-labeled substrates. Proteins 1 and 2 showed high homologies with (1) a β-galactosidase (74%) and (2) a β-xylosidase (84%), whereas the remaining two (5 and 6) were homologous with proteins reported as a diguanylate cyclase and an aquaporin, respectively. The β-galactosidase- and β-xylosidase-like proteins 1 and 2 were confirmed as being responsible for previously found thermo-alkaliphilic glycosidase activities of extracts of E. coli carrying the respective source fosmids. Remarkably, the β-xylosidase-like protein 2 showed activities with both p NP-Xyl and p NP-Ara in the temperature range 40-50 °C and pH range 8.0-10.0. Moreover, proteins 5 and 6 showed thermotolerant α-glucosidase activity at pH 10.0. In silico structure prediction of protein 5 revealed the presence of a potential "GGDEF" catalytic site, encoding α-glucosidase activity, whereas that of protein 6 showed a "GDSL" site, encoding a 'new family' α-glucosidase activity. Using a rational screening approach, we identified and

High Potential Source for Biomass Degradation Enzyme Discovery and Environmental Aspects Revealed through Metagenomics of Indian Buffalo Rumen

Directory of Open Access Journals (Sweden)

K. M. Singh

2014-01-01

Full Text Available The complex microbiomes of the rumen functions as an effective system for plant cell wall degradation, and biomass utilization provide genetic resource for degrading microbial enzymes that could be used in the production of biofuel. Therefore the buffalo rumen microbiota was surveyed using shot gun sequencing. This metagenomic sequencing generated 3.9 GB of sequences and data were assembled into 137270 contiguous sequences (contigs. We identified potential 2614 contigs encoding biomass degrading enzymes including glycoside hydrolases (GH: 1943 contigs, carbohydrate binding module (CBM: 23 contigs, glycosyl transferase (GT: 373 contigs, carbohydrate esterases (CE: 259 contigs, and polysaccharide lyases (PE: 16 contigs. The hierarchical clustering of buffalo metagenomes demonstrated the similarities and dissimilarity in microbial community structures and functional capacity. This demonstrates that buffalo rumen microbiome was considerably enriched in functional genes involved in polysaccharide degradation with great prospects to obtain new molecules that may be applied in the biofuel industry.

Databases of the marine metagenomics

KAUST Repository

Mineta, Katsuhiko

2015-10-28

The metagenomic data obtained from marine environments is significantly useful for understanding marine microbial communities. In comparison with the conventional amplicon-based approach of metagenomics, the recent shotgun sequencing-based approach has become a powerful tool that provides an efficient way of grasping a diversity of the entire microbial community at a sampling point in the sea. However, this approach accelerates accumulation of the metagenome data as well as increase of data complexity. Moreover, when metagenomic approach is used for monitoring a time change of marine environments at multiple locations of the seawater, accumulation of metagenomics data will become tremendous with an enormous speed. Because this kind of situation has started becoming of reality at many marine research institutions and stations all over the world, it looks obvious that the data management and analysis will be confronted by the so-called Big Data issues such as how the database can be constructed in an efficient way and how useful knowledge should be extracted from a vast amount of the data. In this review, we summarize the outline of all the major databases of marine metagenome that are currently publically available, noting that database exclusively on marine metagenome is none but the number of metagenome databases including marine metagenome data are six, unexpectedly still small. We also extend our explanation to the databases, as reference database we call, that will be useful for constructing a marine metagenome database as well as complementing important information with the database. Then, we would point out a number of challenges to be conquered in constructing the marine metagenome database.

In vitro and in silico characterization of metagenomic soil-derived cellulases capable of hydrolyzing oil palm empty fruit bunch

Directory of Open Access Journals (Sweden)

Laura Marcela Palma Medina

2017-09-01

Full Text Available Diversification of raw material for biofuel production is of interest to both academia and industry. One attractive substrate is a renewable lignocellulosic material such as oil palm (Elaeis guineensis Jacq. empty fruit bunch (OPEFB, which is a byproduct of the palm oil industry. This study aimed to characterize cellulases active against this substrate. Cellulases with activity against OPEFB were identified from a metagenomic library obtained from DNA extracted from a high-Andean forest ecosystem. Our findings show that the highest cellulolytic activities were obtained at pH and temperature ranges of 4–10 and 30 °C–60 °C, respectively. Due to the heterogeneous character of the system, degradation profiles were fitted to a fractal-like kinetic model, evidencing transport mass transfer limitations. The sequence analysis of the metagenomic library inserts revealed three glycosyl hydrolase families. Finally, molecular docking simulations of the cellulases were carried out corroborating possible exoglucanase and β-glucosidase activity.

Functional importance of PAI-1 glycosylation

DEFF Research Database (Denmark)

Christensen, Anni; Naessens, Dominik; Skottrup, Peter

2001-01-01

Structure-function studies of plasminogen activator inhibitor-1 (PAI-1) have previously been performed mostly with non-glycosylated material expressed in E. coli. We have now studied the importance of PAI-1 glycosylation for its functional properties. PAI-1 has 3 potential sites for N......-glycosylated PAI-1 could be conferred upon PAI-1 expressed in HEK293 cells by mutational inactivation of one or the other glycosylation site. These findings reveal a novel functional role for glycosylation of a serpin. The glycosylation sites are localised between a-helix H and b-strand 2C and b-strand 3C and a...

Exploration of noncoding sequences in metagenomes.

Directory of Open Access Journals (Sweden)

Fabián Tobar-Tosse

Full Text Available Environment-dependent genomic features have been defined for different metagenomes, whose genes and their associated processes are related to specific environments. Identification of ORFs and their functional categories are the most common methods for association between functional and environmental features. However, this analysis based on finding ORFs misses noncoding sequences and, therefore, some metagenome regulatory or structural information could be discarded. In this work we analyzed 23 whole metagenomes, including coding and noncoding sequences using the following sequence patterns: (G+C content, Codon Usage (Cd, Trinucleotide Usage (Tn, and functional assignments for ORF prediction. Herein, we present evidence of a high proportion of noncoding sequences discarded in common similarity-based methods in metagenomics, and the kind of relevant information present in those. We found a high density of trinucleotide repeat sequences (TRS in noncoding sequences, with a regulatory and adaptive function for metagenome communities. We present associations between trinucleotide values and gene function, where metagenome clustering correlate with microorganism adaptations and kinds of metagenomes. We propose here that noncoding sequences have relevant information to describe metagenomes that could be considered in a whole metagenome analysis in order to improve their organization, classification protocols, and their relation with the environment.

Soil metagenomics and tropical soil productivity

OpenAIRE

Garrett, Karen A.

2009-01-01

This presentation summarizes research in the soil metagenomics cross cutting research activity. Soil metagenomics studies soil microbial communities as contributors to soil health.C CCRA-4 (Soil Metagenomics)

Metagenome Assembly at the DOE JGI (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

Energy Technology Data Exchange (ETDEWEB)

Chain, Patrick

2011-10-13

Patrick Chain of DOE JGI at LANL, Co-Chair of the Metagenome-specific Assembly session, on Metagenome Assembly at the DOE JGIat the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

Exploring neighborhoods in the metagenome universe.

Science.gov (United States)

Aßhauer, Kathrin P; Klingenberg, Heiner; Lingner, Thomas; Meinicke, Peter

2014-07-14

The variety of metagenomes in current databases provides a rapidly growing source of information for comparative studies. However, the quantity and quality of supplementary metadata is still lagging behind. It is therefore important to be able to identify related metagenomes by means of the available sequence data alone. We have studied efficient sequence-based methods for large-scale identification of similar metagenomes within a database retrieval context. In a broad comparison of different profiling methods we found that vector-based distance measures are well-suitable for the detection of metagenomic neighbors. Our evaluation on more than 1700 publicly available metagenomes indicates that for a query metagenome from a particular habitat on average nine out of ten nearest neighbors represent the same habitat category independent of the utilized profiling method or distance measure. While for well-defined labels a neighborhood accuracy of 100% can be achieved, in general the neighbor detection is severely affected by a natural overlap of manually annotated categories. In addition, we present results of a novel visualization method that is able to reflect the similarity of metagenomes in a 2D scatter plot. The visualization method shows a similarly high accuracy in the reduced space as compared with the high-dimensional profile space. Our study suggests that for inspection of metagenome neighborhoods the profiling methods and distance measures can be chosen to provide a convenient interpretation of results in terms of the underlying features. Furthermore, supplementary metadata of metagenome samples in the future needs to comply with readily available ontologies for fine-grained and standardized annotation. To make profile-based k-nearest-neighbor search and the 2D-visualization of the metagenome universe available to the research community, we included the proposed methods in our CoMet-Universe server for comparative metagenome analysis.

Efficient synthesis of glycosylated phenazine natural products and analogs with DISAL (methyl 3,5-dinitrosalicylate) glycosyl donors

DEFF Research Database (Denmark)

Laursen, Jane B.; Petersen, Lars; Jensen, K.J.

2003-01-01

Inspired by the occurrence and function of phenazines in natural products, new glycosylated analogs were designed and synthesized. DISAL (methyl 3,5-dinitrosalicylate) glycosyl donors were used in an efficient and easily-handled glycosylation protocol compatible with combinatorial chemistry....... Benzoylated D-glucose, D-galactose and L-quinovose DISAL glycosyl donors were synthesized in high yields and used under mild conditions to glycosylate methyl saphenate and 2-hydroxyphenazine. The glycosides were screened for biological activity and one compound showed inhibitory activity towards topoisomerase...

Discovery of novel enzymes with industrial potential from a cold and alkaline environment by a combination of functional metagenomics and culturing

DEFF Research Database (Denmark)

Vester, Jan Kjølhede; Glaring, Mikkel Andreas; Stougaard, Peter

2014-01-01

Background: The use of cold-active enzymes has many advantages, including reduced energy consumption and easy inactivation. The ikaite columns of SW Greenland are permanently cold (4-6°C) and alkaline (above pH 10), and the microorganisms living there and their enzymes are adapted to these condit......Background: The use of cold-active enzymes has many advantages, including reduced energy consumption and easy inactivation. The ikaite columns of SW Greenland are permanently cold (4-6°C) and alkaline (above pH 10), and the microorganisms living there and their enzymes are adapted...... to these conditions. Since only a small fraction of the total microbial diversity can be cultured in the laboratory, a combined approach involving functional screening of a strain collection and a metagenomic library was undertaken for discovery of novel enzymes from the ikaite columns.Results: A strain collection...... complemented each other by targeting different microbial communities, highlighting the usefulness of combining methods for bioprospecting. Finally, we document here that ikaite columns constitute an important source of cold- and/or alkaline-active enzymes with industrial application potential....

Bioprospecting thermophiles for cellulase production: a review.

Science.gov (United States)

Acharya, Somen; Chaudhary, Anita

2012-07-01

Most of the potential bioprospecting is currently related to the study of the extremophiles and their potential use in industrial processes. Recently microbial cellulases find applications in various industries and constitute a major group of industrial enzymes. Considerable amount of work has been done on microbial cellulases, especially with resurgence of interest in biomass ethanol production employing cellulases and use of cellulases in textile and paper industry. Most efficient method of lignocellulosic biomass hydrolysis is through enzymatic saccharification using cellulases. Significant information has also been gained about the physiology of thermophilic cellulases producers and process development for enzyme production and biomass saccharification. The review discusses the current knowledge on cellulase producing thermophilic microorganisms, their physiological adaptations and control of cellulase gene expression. It discusses the industrial applications of thermophilic cellulases, their cost of production and challenges in cellulase research especially in the area of improving process economics of enzyme production.

Large-scale bioprospecting of cyanobacteria, micro- and macroalgae from the Aegean Sea

Digital Repository Service at National Institute of Oceanography (India)

Montalvao, S.; Demirel, Z.; PrabhaDevi; Lombardi, V.; Hongisto, V.; Perala, M.; Hattara, J.; Imamoglu, E.; Tilvi, S.; Turan, G.; Dalay, M.C.; Tammela, P.

cells (inhibition of growth 87%) andsimilar effect has previously been reported for this species (Mhadhebi et al. 2014). Recent publications(Mikami and Hosokawa 2013; Mhadhebi et al. 2014) have provided evidence that some compounds and pigments present..., Mathur EJ. Bioprospecting ethics & benefits: a model for effective benefit- sharing. Ind Biotechnol 2005;1:255–9. http://dx.doi.org/ 10.1089/ind.2005.1.255. [2] Beattie AJ, Hay M, Magnusson B, de Nys R, Smeathers J, Vincent JFV. Ecology...

Control of mucin-type O-glycosylation

DEFF Research Database (Denmark)

Bennett, Eric P; Mandel, Ulla; Clausen, Henrik

2012-01-01

residues, is one of the most abundant forms of protein glycosylation in animals. Although most protein glycosylation is controlled by one or two genes encoding the enzymes responsible for the initiation of glycosylation, i.e. the step where the first glycan is attached to the relevant amino acid residue...... in the protein, mucin-type O-glycosylation is controlled by a large family of up to 20 homologous genes encoding UDP-GalNAc:polypeptide GalNAc-transferases (GalNAc-Ts) (EC 2.4.1.41). Therefore, mucin-type O-glycosylation has the greatest potential for differential regulation in cells and tissues. The Gal...

Bioprospecting Archaea: Focus on Extreme Halophiles

KAUST Repository

Antunes, André

2016-12-12

In 1990, Woese et al. divided the Tree of Life into three separate domains: Eukarya, Bacteria, and Archaea. Archaea were originally perceived as little more than “odd bacteria” restricted to extreme environmental niches, but later discoveries challenged this assumption. Members of this domain populate a variety of unexpected environments (e.g. soils, seawater, and human bodies), and we currently witness ongoing massive expansions of the archaeal branch of the Tree of Life. Archaea are now recognized as major players in the biosphere and constitute a significant fraction of the earth’s biomass, yet they remain underexplored. An ongoing surge in exploration efforts is leading to an increase in the (a) number of isolated strains, (b) associated knowledge, and (c) utilization of Archaea in biotechnology. They are increasingly employed in fields as diverse as biocatalysis, biocomputing, bioplastic production, bioremediation, bioengineering, food, pharmaceuticals, and nutraceuticals. This chapter provides a general overview on bioprospecting Archaea, with a particular focus on extreme halophiles. We explore aspects such as diversity, ecology, screening techniques and biotechnology. Current and future trends in mining for applications are discussed.

N- and O-glycosylation Analysis of Human C1-inhibitor Reveals Extensive Mucin-type O-Glycosylation.

Science.gov (United States)

Stavenhagen, Kathrin; Kayili, H Mehmet; Holst, Stephanie; Koeleman, Carolien A M; Engel, Ruchira; Wouters, Diana; Zeerleder, Sacha; Salih, Bekir; Wuhrer, Manfred

2018-06-01

Human C1-inhibitor (C1-Inh) is a serine protease inhibitor and the major regulator of the contact activation pathway as well as the classical and lectin complement pathways. It is known to be a highly glycosylated plasma glycoprotein. However, both the structural features and biological role of C1-Inh glycosylation are largely unknown. Here, we performed for the first time an in-depth site-specific N - and O -glycosylation analysis of C1-Inh combining various mass spectrometric approaches, including C18-porous graphitized carbon (PGC)-LC-ESI-QTOF-MS/MS applying stepping-energy collision-induced dissociation (CID) and electron-transfer dissociation (ETD). Various proteases were applied, partly in combination with PNGase F and exoglycosidase treatment, in order to analyze the (glyco)peptides. The analysis revealed an extensively O -glycosylated N-terminal region. Five novel and five known O -glycosylation sites were identified, carrying mainly core1-type O -glycans. In addition, we detected a heavily O -glycosylated portion spanning from Thr 82 -Ser 121 with up to 16 O -glycans attached. Likewise, all known six N -glycosylation sites were covered and confirmed by this site-specific glycosylation analysis. The glycoforms were in accordance with results on released N -glycans by MALDI-TOF/TOF-MS/MS. The comprehensive characterization of C1-Inh glycosylation described in this study will form the basis for further functional studies on the role of these glycan modifications. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Current and future resources for functional metagenomics

Directory of Open Access Journals (Sweden)

Kathy Nguyen Lam

2015-10-01

Full Text Available Functional metagenomics is a powerful experimental approach for studying gene function, starting from the extracted DNA of mixed microbial populations. A functional approach relies on the construction and screening of metagenomic libraries – physical libraries that contain DNA cloned from environmental metagenomes. The information obtained from functional metagenomics can help in future annotations of gene function and serve as a complement to sequence-based metagenomics. In this Perspective, we begin by summarizing the technical challenges of constructing metagenomic libraries and emphasize their value as resources. We then discuss libraries constructed using the popular cloning vector, pCC1FOS, and highlight the strengths and shortcomings of this system, alongside possible strategies to maximize existing pCC1FOS-based libraries by screening in diverse hosts. Finally, we discuss the known bias of libraries constructed from human gut and marine water samples, present results that suggest bias may also occur for soil libraries, and consider factors that bias metagenomic libraries in general. We anticipate that discussion of current resources and limitations will advance tools and technologies for functional metagenomics research.

Prediction of glycosylation sites using random forests

Directory of Open Access Journals (Sweden)

Hirst Jonathan D

2008-11-01

Full Text Available Abstract Background Post translational modifications (PTMs occur in the vast majority of proteins and are essential for function. Prediction of the sequence location of PTMs enhances the functional characterisation of proteins. Glycosylation is one type of PTM, and is implicated in protein folding, transport and function. Results We use the random forest algorithm and pairwise patterns to predict glycosylation sites. We identify pairwise patterns surrounding glycosylation sites and use an odds ratio to weight their propensity of association with modified residues. Our prediction program, GPP (glycosylation prediction program, predicts glycosylation sites with an accuracy of 90.8% for Ser sites, 92.0% for Thr sites and 92.8% for Asn sites. This is significantly better than current glycosylation predictors. We use the trepan algorithm to extract a set of comprehensible rules from GPP, which provide biological insight into all three major glycosylation types. Conclusion We have created an accurate predictor of glycosylation sites and used this to extract comprehensible rules about the glycosylation process. GPP is available online at http://comp.chem.nottingham.ac.uk/glyco/.

A primer on metagenomics.

Directory of Open Access Journals (Sweden)

John C Wooley

2010-02-01

Full Text Available Metagenomics is a discipline that enables the genomic study of uncultured microorganisms. Faster, cheaper sequencing technologies and the ability to sequence uncultured microbes sampled directly from their habitats are expanding and transforming our view of the microbial world. Distilling meaningful information from the millions of new genomic sequences presents a serious challenge to bioinformaticians. In cultured microbes, the genomic data come from a single clone, making sequence assembly and annotation tractable. In metagenomics, the data come from heterogeneous microbial communities, sometimes containing more than 10,000 species, with the sequence data being noisy and partial. From sampling, to assembly, to gene calling and function prediction, bioinformatics faces new demands in interpreting voluminous, noisy, and often partial sequence data. Although metagenomics is a relative newcomer to science, the past few years have seen an explosion in computational methods applied to metagenomic-based research. It is therefore not within the scope of this article to provide an exhaustive review. Rather, we provide here a concise yet comprehensive introduction to the current computational requirements presented by metagenomics, and review the recent progress made. We also note whether there is software that implements any of the methods presented here, and briefly review its utility. Nevertheless, it would be useful if readers of this article would avail themselves of the comment section provided by this journal, and relate their own experiences. Finally, the last section of this article provides a few representative studies illustrating different facets of recent scientific discoveries made using metagenomics.

Hallmarks of glycosylation in cancer.

Science.gov (United States)

Munkley, Jennifer; Elliott, David J

2016-06-07

Aberrant glycosylation plays a fundamental role in key pathological steps of tumour development and progression. Glycans have roles in cancer cell signalling, tumour cell dissociation and invasion, cell-matrix interactions, angiogenesis, metastasis and immune modulation. Aberrant glycosylation is often cited as a 'hallmark of cancer' but is notably absent from both the original hallmarks of cancer and from the next generation of emerging hallmarks. This review discusses how glycosylation is clearly an enabling characteristic that is causally associated with the acquisition of all the hallmark capabilities. Rather than aberrant glycosylation being itself a hallmark of cancer, another perspective is that glycans play a role in every recognised cancer hallmark.

Bioprospecting thermophiles for cellulase production: a review

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Somen Acharya

2012-09-01

Full Text Available Most of the potential bioprospecting is currently related to the study of the extremophiles and their potential use in industrial processes. Recently microbial cellulases find applications in various industries and constitute a major group of industrial enzymes. Considerable amount of work has been done on microbial cellulases, especially with resurgence of interest in biomass ethanol production employing cellulases and use of cellulases in textile and paper industry. Most efficient method of lignocellulosic biomass hydrolysis is through enzymatic saccharification using cellulases. Significant information has also been gained about the physiology of thermophilic cellulases producers and process development for enzyme production and biomass saccharification. The review discusses the current knowledge on cellulase producing thermophilic microorganisms, their physiological adaptations and control of cellulase gene expression. It discusses the industrial applications of thermophilic cellulases, their cost of production and challenges in cellulase research especially in the area of improving process economics of enzyme production.

Critical Assessment of Metagenome Interpretation – a benchmark of computational metagenomics software

Science.gov (United States)

Sczyrba, Alexander; Hofmann, Peter; Belmann, Peter; Koslicki, David; Janssen, Stefan; Dröge, Johannes; Gregor, Ivan; Majda, Stephan; Fiedler, Jessika; Dahms, Eik; Bremges, Andreas; Fritz, Adrian; Garrido-Oter, Ruben; Jørgensen, Tue Sparholt; Shapiro, Nicole; Blood, Philip D.; Gurevich, Alexey; Bai, Yang; Turaev, Dmitrij; DeMaere, Matthew Z.; Chikhi, Rayan; Nagarajan, Niranjan; Quince, Christopher; Meyer, Fernando; Balvočiūtė, Monika; Hansen, Lars Hestbjerg; Sørensen, Søren J.; Chia, Burton K. H.; Denis, Bertrand; Froula, Jeff L.; Wang, Zhong; Egan, Robert; Kang, Dongwan Don; Cook, Jeffrey J.; Deltel, Charles; Beckstette, Michael; Lemaitre, Claire; Peterlongo, Pierre; Rizk, Guillaume; Lavenier, Dominique; Wu, Yu-Wei; Singer, Steven W.; Jain, Chirag; Strous, Marc; Klingenberg, Heiner; Meinicke, Peter; Barton, Michael; Lingner, Thomas; Lin, Hsin-Hung; Liao, Yu-Chieh; Silva, Genivaldo Gueiros Z.; Cuevas, Daniel A.; Edwards, Robert A.; Saha, Surya; Piro, Vitor C.; Renard, Bernhard Y.; Pop, Mihai; Klenk, Hans-Peter; Göker, Markus; Kyrpides, Nikos C.; Woyke, Tanja; Vorholt, Julia A.; Schulze-Lefert, Paul; Rubin, Edward M.; Darling, Aaron E.; Rattei, Thomas; McHardy, Alice C.

2018-01-01

In metagenome analysis, computational methods for assembly, taxonomic profiling and binning are key components facilitating downstream biological data interpretation. However, a lack of consensus about benchmarking datasets and evaluation metrics complicates proper performance assessment. The Critical Assessment of Metagenome Interpretation (CAMI) challenge has engaged the global developer community to benchmark their programs on datasets of unprecedented complexity and realism. Benchmark metagenomes were generated from ~700 newly sequenced microorganisms and ~600 novel viruses and plasmids, including genomes with varying degrees of relatedness to each other and to publicly available ones and representing common experimental setups. Across all datasets, assembly and genome binning programs performed well for species represented by individual genomes, while performance was substantially affected by the presence of related strains. Taxonomic profiling and binning programs were proficient at high taxonomic ranks, with a notable performance decrease below the family level. Parameter settings substantially impacted performances, underscoring the importance of program reproducibility. While highlighting current challenges in computational metagenomics, the CAMI results provide a roadmap for software selection to answer specific research questions. PMID:28967888

Web Resources for Metagenomics Studies

Directory of Open Access Journals (Sweden)

Pravin Dudhagara

2015-10-01

Full Text Available The development of next-generation sequencing (NGS platforms spawned an enormous volume of data. This explosion in data has unearthed new scalability challenges for existing bioinformatics tools. The analysis of metagenomic sequences using bioinformatics pipelines is complicated by the substantial complexity of these data. In this article, we review several commonly-used online tools for metagenomics data analysis with respect to their quality and detail of analysis using simulated metagenomics data. There are at least a dozen such software tools presently available in the public domain. Among them, MGRAST, IMG/M, and METAVIR are the most well-known tools according to the number of citations by peer-reviewed scientific media up to mid-2015. Here, we describe 12 online tools with respect to their web link, annotation pipelines, clustering methods, online user support, and availability of data storage. We have also done the rating for each tool to screen more potential and preferential tools and evaluated five best tools using synthetic metagenome. The article comprehensively deals with the contemporary problems and the prospects of metagenomics from a bioinformatics viewpoint.

Assembling large, complex environmental metagenomes

Energy Technology Data Exchange (ETDEWEB)

Howe, A. C. [Michigan State Univ., East Lansing, MI (United States). Microbiology and Molecular Genetics, Plant Soil and Microbial Sciences; Jansson, J. [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Earth Sciences Division; Malfatti, S. A. [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Tringe, S. G. [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Tiedje, J. M. [Michigan State Univ., East Lansing, MI (United States). Microbiology and Molecular Genetics, Plant Soil and Microbial Sciences; Brown, C. T. [Michigan State Univ., East Lansing, MI (United States). Microbiology and Molecular Genetics, Computer Science and Engineering

2012-12-28

The large volumes of sequencing data required to sample complex environments deeply pose new challenges to sequence analysis approaches. De novo metagenomic assembly effectively reduces the total amount of data to be analyzed but requires significant computational resources. We apply two pre-assembly filtering approaches, digital normalization and partitioning, to make large metagenome assemblies more computationaly tractable. Using a human gut mock community dataset, we demonstrate that these methods result in assemblies nearly identical to assemblies from unprocessed data. We then assemble two large soil metagenomes from matched Iowa corn and native prairie soils. The predicted functional content and phylogenetic origin of the assembled contigs indicate significant taxonomic differences despite similar function. The assembly strategies presented are generic and can be extended to any metagenome; full source code is freely available under a BSD license.

Trends in the Discovery of New Marine Natural Products from Invertebrates over the Last Two Decades – Where and What Are We Bioprospecting?

Science.gov (United States)

Leal, Miguel Costa; Puga, João; Serôdio, João; Gomes, Newton C. M.; Calado, Ricardo

2012-01-01

It is acknowledged that marine invertebrates produce bioactive natural products that may be useful for developing new drugs. By exploring untapped geographical sources and/or novel groups of organisms one can maximize the search for new marine drugs to treat human diseases. The goal of this paper is to analyse the trends associated with the discovery of new marine natural products from invertebrates (NMNPI) over the last two decades. The analysis considers different taxonomical levels and geographical approaches of bioprospected species. Additionally, this research is also directed to provide new insights into less bioprospected taxa and world regions. In order to gather the information available on NMNPI, the yearly-published reviews of Marine Natural Products covering 1990–2009 were surveyed. Information on source organisms, specifically taxonomical information and collection sites, was assembled together with additional geographical information collected from the articles originally describing the new natural product. Almost 10000 NMNPI were discovered since 1990, with a pronounced increase between decades. Porifera and Cnidaria were the two dominant sources of NMNPI worldwide. The exception was polar regions where Echinodermata dominated. The majority of species that yielded the new natural products belong to only one class of each Porifera and Cnidaria phyla (Demospongiae and Anthozoa, respectively). Increased bioprospecting efforts were observed in the Pacific Ocean, particularly in Asian countries that are associated with the Japan Biodiversity Hotspot and the Kuroshio Current. Although results show comparably less NMNPI from polar regions, the number of new natural products per species is similar to that recorded for other regions. The present study provides information to future bioprospecting efforts addressing previously unexplored taxonomic groups and/or regions. We also highlight how marine invertebrates, which in some cases have no commercial value

Critical Assessment of Metagenome Interpretation

DEFF Research Database (Denmark)

Sczyrba, Alexander; Hofmann, Peter; Belmann, Peter

2017-01-01

Methods for assembly, taxonomic profiling and binning are key to interpreting metagenome data, but a lack of consensus about benchmarking complicates performance assessment. The Critical Assessment of Metagenome Interpretation (CAMI) challenge has engaged the global developer community to benchma...

Functional importance of PAI-1 glycosylation

DEFF Research Database (Denmark)

Christensen, Anni; Naessens, Dominik; Skottrup, Peter

susceptible PAI-1 variant was not necessarily the one used when raising the antibody. This and other observations indicated that the carbohydrate moieties or the glycosylation sites are unlikely to be part of the epitopes for these antibodies. The antibody susceptibility characteristic for non......Structure-function studies of plasminogen activator inhibitor-1 (PAI-1) have previously been performed mostly with non-glycosylated material expressed in E. coli. We have now studied the importance of PAI-1 glycosylation for its functional properties. PAI-1 has 3 potential sites for N......-linked glycosylation. Biochemical analysis of PAI-1 variants with substitutions of the Asn residues in each of these sites and expression in human embryonic kidney 293 (HEK293) cells showed that only Asn211 and Asn 267, but not Asn331 are glycosylated, and revealed a differential composition of the carbohydrate...

Venom evolution widespread in fishes: a phylogenetic road map for the bioprospecting of piscine venoms.

Science.gov (United States)

Smith, William Leo; Wheeler, Ward C

2006-01-01

Knowledge of evolutionary relationships or phylogeny allows for effective predictions about the unstudied characteristics of species. These include the presence and biological activity of an organism's venoms. To date, most venom bioprospecting has focused on snakes, resulting in six stroke and cancer treatment drugs that are nearing U.S. Food and Drug Administration review. Fishes, however, with thousands of venoms, represent an untapped resource of natural products. The first step involved in the efficient bioprospecting of these compounds is a phylogeny of venomous fishes. Here, we show the results of such an analysis and provide the first explicit suborder-level phylogeny for spiny-rayed fishes. The results, based on approximately 1.1 million aligned base pairs, suggest that, in contrast to previous estimates of 200 venomous fishes, >1,200 fishes in 12 clades should be presumed venomous. This assertion was corroborated by a detailed anatomical study examining potentially venomous structures in >100 species. The results of these studies not only alter our view of the diversity of venomous fishes, now representing >50% of venomous vertebrates, but also provide the predictive phylogeny or "road map" for the efficient search for potential pharmacological agents or physiological tools from the unexplored fish venoms.

Preliminary High-Throughput Metagenome Assembly

Energy Technology Data Exchange (ETDEWEB)

Dusheyko, Serge; Furman, Craig; Pangilinan, Jasmyn; Shapiro, Harris; Tu, Hank

2007-03-26

Metagenome data sets present a qualitatively different assembly problem than traditional single-organism whole-genome shotgun (WGS) assembly. The unique aspects of such projects include the presence of a potentially large number of distinct organisms and their representation in the data set at widely different fractions. In addition, multiple closely related strains could be present, which would be difficult to assemble separately. Failure to take these issues into account can result in poor assemblies that either jumble together different strains or which fail to yield useful results. The DOE Joint Genome Institute has sequenced a number of metagenomic projects and plans to considerably increase this number in the coming year. As a result, the JGI has a need for high-throughput tools and techniques for handling metagenome projects. We present the techniques developed to handle metagenome assemblies in a high-throughput environment. This includes a streamlined assembly wrapper, based on the JGI?s in-house WGS assembler, Jazz. It also includes the selection of sensible defaults targeted for metagenome data sets, as well as quality control automation for cleaning up the raw results. While analysis is ongoing, we will discuss preliminary assessments of the quality of the assembly results (http://fames.jgi-psf.org).

Viral Metagenomics: MetaView Software

Energy Technology Data Exchange (ETDEWEB)

Zhou, C; Smith, J

2007-10-22

The purpose of this report is to design and develop a tool for analysis of raw sequence read data from viral metagenomics experiments. The tool should compare read sequences of known viral nucleic acid sequence data and enable a user to attempt to determine, with some degree of confidence, what virus groups may be present in the sample. This project was conducted in two phases. In phase 1 we surveyed the literature and examined existing metagenomics tools to educate ourselves and to more precisely define the problem of analyzing raw read data from viral metagenomic experiments. In phase 2 we devised an approach and built a prototype code and database. This code takes viral metagenomic read data in fasta format as input and accesses all complete viral genomes from Kpath for sequence comparison. The system executes at the UNIX command line, producing output that is stored in an Oracle relational database. We provide here a description of the approach we came up with for handling un-assembled, short read data sets from viral metagenomics experiments. We include a discussion of the current MetaView code capabilities and additional functionality that we believe should be added, should additional funding be acquired to continue the work.

Beyond biodiversity: fish metagenomes.

Science.gov (United States)

Ardura, Alba; Planes, Serge; Garcia-Vazquez, Eva

2011-01-01

Biodiversity and intra-specific genetic diversity are interrelated and determine the potential of a community to survive and evolve. Both are considered together in Prokaryote communities treated as metagenomes or ensembles of functional variants beyond species limits.Many factors alter biodiversity in higher Eukaryote communities, and human exploitation can be one of the most important for some groups of plants and animals. For example, fisheries can modify both biodiversity and genetic diversity (intra specific). Intra-specific diversity can be drastically altered by overfishing. Intense fishing pressure on one stock may imply extinction of some genetic variants and subsequent loss of intra-specific diversity. The objective of this study was to apply a metagenome approach to fish communities and explore its value for rapid evaluation of biodiversity and genetic diversity at community level. Here we have applied the metagenome approach employing the barcoding target gene coi as a model sequence in catch from four very different fish assemblages exploited by fisheries: freshwater communities from the Amazon River and northern Spanish rivers, and marine communities from the Cantabric and Mediterranean seas.Treating all sequences obtained from each regional catch as a biological unit (exploited community) we found that metagenomic diversity indices of the Amazonian catch sample here examined were lower than expected. Reduced diversity could be explained, at least partially, by overexploitation of the fish community that had been independently estimated by other methods.We propose using a metagenome approach for estimating diversity in Eukaryote communities and early evaluating genetic variation losses at multi-species level.

Beyond biodiversity: fish metagenomes.

Directory of Open Access Journals (Sweden)

Alba Ardura

Full Text Available Biodiversity and intra-specific genetic diversity are interrelated and determine the potential of a community to survive and evolve. Both are considered together in Prokaryote communities treated as metagenomes or ensembles of functional variants beyond species limits.Many factors alter biodiversity in higher Eukaryote communities, and human exploitation can be one of the most important for some groups of plants and animals. For example, fisheries can modify both biodiversity and genetic diversity (intra specific. Intra-specific diversity can be drastically altered by overfishing. Intense fishing pressure on one stock may imply extinction of some genetic variants and subsequent loss of intra-specific diversity. The objective of this study was to apply a metagenome approach to fish communities and explore its value for rapid evaluation of biodiversity and genetic diversity at community level. Here we have applied the metagenome approach employing the barcoding target gene coi as a model sequence in catch from four very different fish assemblages exploited by fisheries: freshwater communities from the Amazon River and northern Spanish rivers, and marine communities from the Cantabric and Mediterranean seas.Treating all sequences obtained from each regional catch as a biological unit (exploited community we found that metagenomic diversity indices of the Amazonian catch sample here examined were lower than expected. Reduced diversity could be explained, at least partially, by overexploitation of the fish community that had been independently estimated by other methods.We propose using a metagenome approach for estimating diversity in Eukaryote communities and early evaluating genetic variation losses at multi-species level.

Gene Prediction in Metagenomic Fragments with Deep Learning

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Shao-Wu Zhang

2017-01-01

Full Text Available Next generation sequencing technologies used in metagenomics yield numerous sequencing fragments which come from thousands of different species. Accurately identifying genes from metagenomics fragments is one of the most fundamental issues in metagenomics. In this article, by fusing multifeatures (i.e., monocodon usage, monoamino acid usage, ORF length coverage, and Z-curve features and using deep stacking networks learning model, we present a novel method (called Meta-MFDL to predict the metagenomic genes. The results with 10 CV and independent tests show that Meta-MFDL is a powerful tool for identifying genes from metagenomic fragments.

Human milk metagenome: a functional capacity analysis

Science.gov (United States)

2013-01-01

Background Human milk contains a diverse population of bacteria that likely influences colonization of the infant gastrointestinal tract. Recent studies, however, have been limited to characterization of this microbial community by 16S rRNA analysis. In the present study, a metagenomic approach using Illumina sequencing of a pooled milk sample (ten donors) was employed to determine the genera of bacteria and the types of bacterial open reading frames in human milk that may influence bacterial establishment and stability in this primal food matrix. The human milk metagenome was also compared to that of breast-fed and formula-fed infants’ feces (n = 5, each) and mothers’ feces (n = 3) at the phylum level and at a functional level using open reading frame abundance. Additionally, immune-modulatory bacterial-DNA motifs were also searched for within human milk. Results The bacterial community in human milk contained over 360 prokaryotic genera, with sequences aligning predominantly to the phyla of Proteobacteria (65%) and Firmicutes (34%), and the genera of Pseudomonas (61.1%), Staphylococcus (33.4%) and Streptococcus (0.5%). From assembled human milk-derived contigs, 30,128 open reading frames were annotated and assigned to functional categories. When compared to the metagenome of infants’ and mothers’ feces, the human milk metagenome was less diverse at the phylum level, and contained more open reading frames associated with nitrogen metabolism, membrane transport and stress response (P milk metagenome also contained a similar occurrence of immune-modulatory DNA motifs to that of infants’ and mothers’ fecal metagenomes. Conclusions Our results further expand the complexity of the human milk metagenome and enforce the benefits of human milk ingestion on the microbial colonization of the infant gut and immunity. Discovery of immune-modulatory motifs in the metagenome of human milk indicates more exhaustive analyses of the functionality of the human

Engineering Mammalian Mucin-type O-Glycosylation in Plants

DEFF Research Database (Denmark)

Yang, Zhang; Drew, Damian P; Jørgensen, Bodil

2012-01-01

-glycans are attached to proteins, and which structures are formed, difficult. Because plants are devoid of GalNAc-type O-glycosylation, we have assessed requirements for establishing human GalNAc O-glycosylation de novo in plants with the aim of developing cell systems with custom-designed O-glycosylation capacity...... was glycosylated with up to three and five GalNAc residues when co-expressed with GalNAc-T2 and a combination of GalNAc-T2 and GalNAc-T4, respectively, as determined by mass spectrometry. O-Glycosylation was furthermore demonstrated on a tandem repeat of MUC16 and interferon a2b. In plants, prolines in certain...... classes of proteins are hydroxylated and further substituted with plant-specific O-glycosylation; unsubstituted hydroxyprolines were identified in our MUC1 construct. In summary, this study demonstrates that mammalian type O-glycosylation can be established in plants and that plants may serve as a host...

Trans-species Engineering of Glycosylated Therapeutic Proteins

DEFF Research Database (Denmark)

Yang, Zhang

important to address. Whenever glycosylation has been found to be an important PTM for function or bioactivity, human therapeutics have generally been produced in mammalian Chinese hamster ovary (CHO) cell line. Oglycosylation is one of the most complex regulated PTMs of proteins but also one of the least...... understood. Currently, mammalian cells are required for human O-glycosylation. Increasing efforts have been devoted to engineering non-mammalian cells for production of recombinant proteins with “human-like” glycosylation. Substantial success has been achieved with designed N-glycosylation in both lower......Recombinant expression of therapeutic proteins is one of the major tasks in modern biomedicine. One of the most important factors with respect to therapeutic use in human is posttranslational modifications (PTMs) of the recombinant proteins, of which protein glycosylation is by far the most...

A Bioinformatician's Guide to Metagenomics

Energy Technology Data Exchange (ETDEWEB)

Kunin, Victor; Copeland, Alex; Lapidus, Alla; Mavromatis, Konstantinos; Hugenholtz, Philip

2008-08-01

As random shotgun metagenomic projects proliferate and become the dominant source of publicly available sequence data, procedures for best practices in their execution and analysis become increasingly important. Based on our experience at the Joint Genome Institute, we describe step-by-step the chain of decisions accompanying a metagenomic project from the viewpoint of a bioinformatician. We guide the reader through a standard workflow for a metagenomic project beginning with pre-sequencing considerations such as community composition and sequence data type that will greatly influence downstream analyses. We proceed with recommendations for sampling and data generation including sample and metadata collection, community profiling, construction of shotgun libraries and sequencing strategies. We then discuss the application of generic sequence processing steps (read preprocessing, assembly, and gene prediction and annotation) to metagenomic datasets by contrast to genome projects. Different types of data analyses particular to metagenomes are then presented including binning, dominant population analysis and gene-centric analysis. Finally data management systems and issues are presented and discussed. We hope that this review will assist bioinformaticians and biologists in making better-informed decisions on their journey during a metagenomic project.

Discovery of novel enzymes with industrial potential from a cold and alkaline environment by a combination of functional metagenomics and culturing.

Science.gov (United States)

Vester, Jan Kjølhede; Glaring, Mikkel Andreas; Stougaard, Peter

2014-05-20

The use of cold-active enzymes has many advantages, including reduced energy consumption and easy inactivation. The ikaite columns of SW Greenland are permanently cold (4-6°C) and alkaline (above pH 10), and the microorganisms living there and their enzymes are adapted to these conditions. Since only a small fraction of the total microbial diversity can be cultured in the laboratory, a combined approach involving functional screening of a strain collection and a metagenomic library was undertaken for discovery of novel enzymes from the ikaite columns. A strain collection with 322 cultured isolates was screened for enzymatic activities identifying a large number of enzyme producers, with a high re-discovery rate to previously characterized strains. A functional expression library established in Escherichia coli identified a number of novel cold-active enzymes. Both α-amylases and β-galactosidases were characterized in more detail with respect to temperature and pH profiles and one of the β-galactosidases, BGalI17E2, was able to hydrolyze lactose at 5°C. A metagenome sequence of the expression library indicated that the majority of enzymatic activities were not detected by functional expression. Phylogenetic analysis showed that different bacterial communities were targeted with the culture dependent and independent approaches and revealed the bias of multiple displacement amplification (MDA) of DNA isolated from complex microbial communities. Many cold- and/or alkaline-active enzymes of industrial relevance were identified in the culture based approach and the majority of the enzyme-producing isolates were closely related to previously characterized strains. The function-based metagenomic approach, on the other hand, identified several enzymes (β-galactosidases, α-amylases and a phosphatase) with low homology to known sequences that were easily expressed in the production host E. coli. The β-galactosidase BGalI17E2 was able to hydrolyze lactose at low

Challenges and Opportunities of Airborne Metagenomics

KAUST Repository

Behzad, H.

2015-05-06

Recent metagenomic studies of environments, such as marine and soil, have significantly enhanced our understanding of the diverse microbial communities living in these habitats and their essential roles in sustaining vast ecosystems. The increase in the number of publications related to soil and marine metagenomics is in sharp contrast to those of air, yet airborne microbes are thought to have significant impacts on many aspects of our lives from their potential roles in atmospheric events such as cloud formation, precipitation, and atmospheric chemistry to their major impact on human health. In this review, we will discuss the current progress in airborne metagenomics, with a special focus on exploring the challenges and opportunities of undertaking such studies. The main challenges of conducting metagenomic studies of airborne microbes are as follows: 1) Low density of microorganisms in the air, 2) efficient retrieval of microorganisms from the air, 3) variability in airborne microbial community composition, 4) the lack of standardized protocols and methodologies, and 5) DNA sequencing and bioinformatics-related challenges. Overcoming these challenges could provide the groundwork for comprehensive analysis of airborne microbes and their potential impact on the atmosphere, global climate, and our health. Metagenomic studies offer a unique opportunity to examine viral and bacterial diversity in the air and monitor their spread locally or across the globe, including threats from pathogenic microorganisms. Airborne metagenomic studies could also lead to discoveries of novel genes and metabolic pathways relevant to meteorological and industrial applications, environmental bioremediation, and biogeochemical cycles.

NeSSM: a Next-generation Sequencing Simulator for Metagenomics.

Directory of Open Access Journals (Sweden)

Ben Jia

Full Text Available BACKGROUND: Metagenomics can reveal the vast majority of microbes that have been missed by traditional cultivation-based methods. Due to its extremely wide range of application areas, fast metagenome sequencing simulation systems with high fidelity are in great demand to facilitate the development and comparison of metagenomics analysis tools. RESULTS: We present here a customizable metagenome simulation system: NeSSM (Next-generation Sequencing Simulator for Metagenomics. Combining complete genomes currently available, a community composition table, and sequencing parameters, it can simulate metagenome sequencing better than existing systems. Sequencing error models based on the explicit distribution of errors at each base and sequencing coverage bias are incorporated in the simulation. In order to improve the fidelity of simulation, tools are provided by NeSSM to estimate the sequencing error models, sequencing coverage bias and the community composition directly from existing metagenome sequencing data. Currently, NeSSM supports single-end and pair-end sequencing for both 454 and Illumina platforms. In addition, a GPU (graphics processing units version of NeSSM is also developed to accelerate the simulation. By comparing the simulated sequencing data from NeSSM with experimental metagenome sequencing data, we have demonstrated that NeSSM performs better in many aspects than existing popular metagenome simulators, such as MetaSim, GemSIM and Grinder. The GPU version of NeSSM is more than one-order of magnitude faster than MetaSim. CONCLUSIONS: NeSSM is a fast simulation system for high-throughput metagenome sequencing. It can be helpful to develop tools and evaluate strategies for metagenomics analysis and it's freely available for academic users at http://cbb.sjtu.edu.cn/~ccwei/pub/software/NeSSM.php.

Comparative metagenomics of the Red Sea

KAUST Repository

Mineta, Katsuhiko

2016-01-26

Metagenome produces a tremendous amount of data that comes from the organisms living in the environments. This big data enables us to examine not only microbial genes but also the community structure, interaction and adaptation mechanisms at the specific location and condition. The Red Sea has several unique characteristics such as high salinity, high temperature and low nutrition. These features must contribute to form the unique microbial community during the evolutionary process. Since 2014, we started monthly samplings of the metagenomes in the Red Sea under KAUST-CCF project. In collaboration with Kitasato University, we also collected the metagenome data from the ocean in Japan, which shows contrasting features to the Red Sea. Therefore, the comparative metagenomics of those data provides a comprehensive view of the Red Sea microbes, leading to identify key microbes, genes and networks related to those environmental differences.

Comparative metagenomics of the Red Sea

KAUST Repository

Mineta, Katsuhiko

2016-01-01

started monthly samplings of the metagenomes in the Red Sea under KAUST-CCF project. In collaboration with Kitasato University, we also collected the metagenome data from the ocean in Japan, which shows contrasting features to the Red Sea. Therefore

Metagenomic applications in environmental monitoring and bioremediation.

Science.gov (United States)

Techtmann, Stephen M; Hazen, Terry C

2016-10-01

With the rapid advances in sequencing technology, the cost of sequencing has dramatically dropped and the scale of sequencing projects has increased accordingly. This has provided the opportunity for the routine use of sequencing techniques in the monitoring of environmental microbes. While metagenomic applications have been routinely applied to better understand the ecology and diversity of microbes, their use in environmental monitoring and bioremediation is increasingly common. In this review we seek to provide an overview of some of the metagenomic techniques used in environmental systems biology, addressing their application and limitation. We will also provide several recent examples of the application of metagenomics to bioremediation. We discuss examples where microbial communities have been used to predict the presence and extent of contamination, examples of how metagenomics can be used to characterize the process of natural attenuation by unculturable microbes, as well as examples detailing the use of metagenomics to understand the impact of biostimulation on microbial communities.

Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments.

Science.gov (United States)

Yamakoshi, Yasuo; Nagano, Takatoshi; Hu, Jan Cc; Yamakoshi, Fumiko; Simmer, James P

2011-02-03

Dentin sialophosphoprotein (Dspp) is a multidomain, secreted protein that is critical for the formation of tooth dentin. Mutations in DSPP cause inherited dentin defects categorized as dentin dysplasia type II and dentinogenesis imperfecta type II and type III. Dentin sialoprotein (Dsp), the N-terminal domain of dentin sialophosphoprotein (Dspp), is a highly glycosylated proteoglycan, but little is known about the number, character, and attachment sites of its carbohydrate moieties. To identify its carbohydrate attachment sites we isolated Dsp from developing porcine molars and digested it with endoproteinase Glu-C or pronase, fractionated the digestion products, identified fractions containing glycosylated peptides using a phenol sulfuric acid assay, and characterized the glycopeptides by N-terminal sequencing, amino acid analyses, or LC/MSMS. To determine the average number of sialic acid attachments per N-glycosylation, we digested Dsp with glycopeptidase A, labeled the released N-glycosylations with 2-aminobenzoic acid, and quantified the moles of released glycosylations by comparison to labeled standards of known concentration. Sialic acid was released by sialidase digestion and quantified by measuring β-NADH reduction of pyruvic acid, which was generated stoichiometrically from sialic acid by aldolase. To determine its forms, sialic acid released by sialidase digestion was labeled with 1,2-diamino-4,5-methyleneoxybenzene (DMB) and compared to a DMB-labeled sialic acid reference panel by RP-HPLC. To determine the composition of Dsp glycosaminoglycan (GAG) attachments, we digested Dsp with chondroitinase ABC and compared the chromotagraphic profiles of the released disaccharides to commercial standards. N-glycosylations were identified at Asn37, Asn77, Asn136, Asn155, Asn161, and Asn176. Dsp averages one sialic acid per N-glycosylation, which is always in the form of N-acetylneuraminic acid. O-glycosylations were tentatively assigned at Thr200, Thr216 and Thr

Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments

Directory of Open Access Journals (Sweden)

Yamakoshi Fumiko

2011-02-01

Full Text Available Abstract Background Dentin sialophosphoprotein (Dspp is a multidomain, secreted protein that is critical for the formation of tooth dentin. Mutations in DSPP cause inherited dentin defects categorized as dentin dysplasia type II and dentinogenesis imperfecta type II and type III. Dentin sialoprotein (Dsp, the N-terminal domain of dentin sialophosphoprotein (Dspp, is a highly glycosylated proteoglycan, but little is known about the number, character, and attachment sites of its carbohydrate moieties. Results To identify its carbohydrate attachment sites we isolated Dsp from developing porcine molars and digested it with endoproteinase Glu-C or pronase, fractionated the digestion products, identified fractions containing glycosylated peptides using a phenol sulfuric acid assay, and characterized the glycopeptides by N-terminal sequencing, amino acid analyses, or LC/MSMS. To determine the average number of sialic acid attachments per N-glycosylation, we digested Dsp with glycopeptidase A, labeled the released N-glycosylations with 2-aminobenzoic acid, and quantified the moles of released glycosylations by comparison to labeled standards of known concentration. Sialic acid was released by sialidase digestion and quantified by measuring β-NADH reduction of pyruvic acid, which was generated stoichiometrically from sialic acid by aldolase. To determine its forms, sialic acid released by sialidase digestion was labeled with 1,2-diamino-4,5-methyleneoxybenzene (DMB and compared to a DMB-labeled sialic acid reference panel by RP-HPLC. To determine the composition of Dsp glycosaminoglycan (GAG attachments, we digested Dsp with chondroitinase ABC and compared the chromotagraphic profiles of the released disaccharides to commercial standards. N-glycosylations were identified at Asn37, Asn77, Asn136, Asn155, Asn161, and Asn176. Dsp averages one sialic acid per N-glycosylation, which is always in the form of N-acetylneuraminic acid. O-glycosylations were

Metagenomic analysis of microbial communities and beyond

DEFF Research Database (Denmark)

Schreiber, Lars

2014-01-01

From small clone libraries to large next-generation sequencing datasets – the field of community genomics or metagenomics has developed tremendously within the last years. This chapter will summarize some of these developments and will also highlight pitfalls of current metagenomic analyses...... heterologous expression of metagenomic DNA fragments to discover novel metabolic functions. Lastly, the chapter will shortly discuss the meta-analysis of gene expression of microbial communities, more precisely metatranscriptomics and metaproteomics....

Biochemical Importance of Glycosylation of Plasminogen Activator Inhibitor-1

DEFF Research Database (Denmark)

Gils, Ann; Pedersen, Katrine Egelund; Skottrup, Peter

2003-01-01

The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous glycosyla......The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous...... with the glycosylation sites could be excluded as explanation for the differential reactivity. The latency transition of non-glycosylated, but not of glycosylated PAI-1, was strongly accelerated by a non-ionic detergent. The different biochemical properties of glycosylated and non-glycosylated PAI-1 depended...

Interactive metagenomic visualization in a Web browser

Directory of Open Access Journals (Sweden)

Phillippy Adam M

2011-09-01

Full Text Available Abstract Background A critical output of metagenomic studies is the estimation of abundances of taxonomical or functional groups. The inherent uncertainty in assignments to these groups makes it important to consider both their hierarchical contexts and their prediction confidence. The current tools for visualizing metagenomic data, however, omit or distort quantitative hierarchical relationships and lack the facility for displaying secondary variables. Results Here we present Krona, a new visualization tool that allows intuitive exploration of relative abundances and confidences within the complex hierarchies of metagenomic classifications. Krona combines a variant of radial, space-filling displays with parametric coloring and interactive polar-coordinate zooming. The HTML5 and JavaScript implementation enables fully interactive charts that can be explored with any modern Web browser, without the need for installed software or plug-ins. This Web-based architecture also allows each chart to be an independent document, making them easy to share via e-mail or post to a standard Web server. To illustrate Krona's utility, we describe its application to various metagenomic data sets and its compatibility with popular metagenomic analysis tools. Conclusions Krona is both a powerful metagenomic visualization tool and a demonstration of the potential of HTML5 for highly accessible bioinformatic visualizations. Its rich and interactive displays facilitate more informed interpretations of metagenomic analyses, while its implementation as a browser-based application makes it extremely portable and easily adopted into existing analysis packages. Both the Krona rendering code and conversion tools are freely available under a BSD open-source license, and available from: http://krona.sourceforge.net.

Glycosylation: a hallmark of cancer?

Science.gov (United States)

Vajaria, Bhairavi N; Patel, Prabhudas S

2017-04-01

The hallmarks of cancer are characterized by functional capabilities that allow cancer cells to survive, proliferate and disseminate during the multistep tumorigenesis. Cancer being a cellular disease, changes in cellular glycoproteins play an important role in malignant transformation and cancer progression. The present review summarizes various studies that depicted correlation of glycosylation with tumor initiation, progression and metastasis, which are helpful in early diagnosis, disease monitoring and prognosis. The results are further strengthened by our reports, which depicted alterations in sialylation and fucosylation in different cancers. Alterations in glycosyltransferases are also involved in formation of various tumor antigens (e.g. Sialyl Lewis x) which serves as ligand for the cell adhesion molecule, selectin which is involved in adhesion of cancer cells to vascular endothelium and thus contributes to hematogenous metastasis. Increased glycosylation accompanied by alterations in glycosyltranferases, glycosidases, glycans and mucins (MUC)s are also involved in loss of E-cadherin, a key molecule implicated in metastatic dissemination of cells. The present review also summarizes the correlation of glycosylation with all the hallmarks of cancer. The enormous progress in the design of novel inhibitors of pathway intermediates of sialylation and fucosylation can prove wonders in combating the dreadful disease. The results provide the evidence that altered glycosylation is linked to tumor initiation, progression and metastasis. Hence, it can be considered as a new hallmark of cancer development and strategies to develop novel glycosylation targeted molecules should be strengthened.

Single Cell and Metagenomic Assemblies: Biology Drives Technical Choices and Goals (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

Energy Technology Data Exchange (ETDEWEB)

Stepanauskas, Ramunas

2011-10-13

DOE JGI's Tanja Woyke, chair of the Single Cells and Metagenomes session, delivers an introduction, followed by Bigelow Laboratory's Ramunas Stepanauskas on "Single Cell and Metagenomic Assemblies: Biology Drives Technical Choices and Goals" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

An Experimental Metagenome Data Management and AnalysisSystem

Energy Technology Data Exchange (ETDEWEB)

Markowitz, Victor M.; Korzeniewski, Frank; Palaniappan, Krishna; Szeto, Ernest; Ivanova, Natalia N.; Kyrpides, Nikos C.; Hugenholtz, Philip

2006-03-01

The application of shotgun sequencing to environmental samples has revealed a new universe of microbial community genomes (metagenomes) involving previously uncultured organisms. Metagenome analysis, which is expected to provide a comprehensive picture of the gene functions and metabolic capacity of microbial community, needs to be conducted in the context of a comprehensive data management and analysis system. We present in this paper IMG/M, an experimental metagenome data management and analysis system that is based on the Integrated Microbial Genomes (IMG) system. IMG/M provides tools and viewers for analyzing both metagenomes and isolate genomes individually or in a comparative context.

Shotgun metagenomic data streams: surfing without fear

Energy Technology Data Exchange (ETDEWEB)

Berendzen, Joel R [Los Alamos National Laboratory

2010-12-06

Timely information about bio-threat prevalence, consequence, propagation, attribution, and mitigation is needed to support decision-making, both routinely and in a crisis. One DNA sequencer can stream 25 Gbp of information per day, but sampling strategies and analysis techniques are needed to turn raw sequencing power into actionable knowledge. Shotgun metagenomics can enable biosurveillance at the level of a single city, hospital, or airplane. Metagenomics characterizes viruses and bacteria from complex environments such as soil, air filters, or sewage. Unlike targeted-primer-based sequencing, shotgun methods are not blind to sequences that are truly novel, and they can measure absolute prevalence. Shotgun metagenomic sampling can be non-invasive, efficient, and inexpensive while being informative. We have developed analysis techniques for shotgun metagenomic sequencing that rely upon phylogenetic signature patterns. They work by indexing local sequence patterns in a manner similar to web search engines. Our methods are laptop-fast and favorable scaling properties ensure they will be sustainable as sequencing methods grow. We show examples of application to soil metagenomic samples.

Competition between folding and glycosylation in the endoplasmic reticulum

DEFF Research Database (Denmark)

Holst, B; Bruun, A W; Kielland-Brandt, Morten

1996-01-01

Using carboxypeptidase Y in Saccharomyces cerevisiae as a model system, the in vivo relationship between protein folding and N-glycosylation was studied. Seven new sites for N-glycosylation were introduced at positions buried in the folded protein structure. The level of glycosylation of such new...... acceptor sites. In some cases, all the newly synthesized mutant protein was modified at the novel site while in others no modification took place. In the most interesting category of mutants, the level of glycosylation was dependent on the conditions for folding. This shows that folding and glycosylation...

Rapid and efficient method to extract metagenomic DNA from estuarine sediments.

Science.gov (United States)

Shamim, Kashif; Sharma, Jaya; Dubey, Santosh Kumar

2017-07-01

Metagenomic DNA from sediments of selective estuaries of Goa, India was extracted using a simple, fast, efficient and environment friendly method. The recovery of pure metagenomic DNA from our method was significantly high as compared to other well-known methods since the concentration of recovered metagenomic DNA ranged from 1185.1 to 4579.7 µg/g of sediment. The purity of metagenomic DNA was also considerably high as the ratio of absorbance at 260 and 280 nm ranged from 1.88 to 1.94. Therefore, the recovered metagenomic DNA was directly used to perform various molecular biology experiments viz. restriction digestion, PCR amplification, cloning and metagenomic library construction. This clearly proved that our protocol for metagenomic DNA extraction using silica gel efficiently removed the contaminants and prevented shearing of the metagenomic DNA. Thus, this modified method can be used to recover pure metagenomic DNA from various estuarine sediments in a rapid, efficient and eco-friendly manner.

Bioprospecting of antimicrobial activity of extracts of endophytic fungi from Bauhinia guianensis.

Science.gov (United States)

Pinheiro, Eduardo A A; Pina, Jeferson R S; Feitosa, André O; Carvalho, Josiwander M; Borges, Fábio C; Marinho, Patrícia S B; Marinho, Andrey M R

Antibiotic resistance results in higher medical costs, prolonged hospital stays and increased mortality and is rising to dangerously high levels in all parts of the world. Therefore, this study aims to search for new antimicrobial agents through bioprospecting of extracts of endophytic fungi from Bauhinia guianensis, a typical Amazonian plant used in combating infections. Seventeen (17) fungi were isolated and as result the methanolic extract of the fungus Exserohilum rostratum showed good activity against the bacteria tested. The polyketide monocerin was isolated by the chromatographic technique, identified by NMR and MS, showing broad antimicrobial spectrum. Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Challenges and opportunities of airborne metagenomics.

Science.gov (United States)

Behzad, Hayedeh; Gojobori, Takashi; Mineta, Katsuhiko

2015-05-06

Recent metagenomic studies of environments, such as marine and soil, have significantly enhanced our understanding of the diverse microbial communities living in these habitats and their essential roles in sustaining vast ecosystems. The increase in the number of publications related to soil and marine metagenomics is in sharp contrast to those of air, yet airborne microbes are thought to have significant impacts on many aspects of our lives from their potential roles in atmospheric events such as cloud formation, precipitation, and atmospheric chemistry to their major impact on human health. In this review, we will discuss the current progress in airborne metagenomics, with a special focus on exploring the challenges and opportunities of undertaking such studies. The main challenges of conducting metagenomic studies of airborne microbes are as follows: 1) Low density of microorganisms in the air, 2) efficient retrieval of microorganisms from the air, 3) variability in airborne microbial community composition, 4) the lack of standardized protocols and methodologies, and 5) DNA sequencing and bioinformatics-related challenges. Overcoming these challenges could provide the groundwork for comprehensive analysis of airborne microbes and their potential impact on the atmosphere, global climate, and our health. Metagenomic studies offer a unique opportunity to examine viral and bacterial diversity in the air and monitor their spread locally or across the globe, including threats from pathogenic microorganisms. Airborne metagenomic studies could also lead to discoveries of novel genes and metabolic pathways relevant to meteorological and industrial applications, environmental bioremediation, and biogeochemical cycles. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Defectively N-glycosylated and non-O-glycosylated aminopeptidase N (CD13) is normally expressed at the cell surface and has full enzymatic activity

DEFF Research Database (Denmark)

Norén, K; Hansen, Gert Helge; Clausen, H

1997-01-01

In order to study the effects of the absence of O-glycosylation and modifications of N-glycosylation on a class II membrane protein, pig and human aminopeptidase N (CD13) were stably expressed in the ldl(D) cell line. This cell line carries a UDP-Gal/UDP-GalNAc-epimerase deficiency which blocks...... the conversion of glucose into galactose derivatives. Thus it is possible in the ldl(D) cell line to selectively block O-glycosylation by the omission of N-acetylgalactoseamine from the culture medium and to alter N-glycosylation by the omission of galactose. In this way selectively altered glycosylated forms...

Indicadores ibero-americanos de atividade científica em bioprospecçãoIndicators of scientific activity in bioprospecting in the ibero-american geopolitical region

Directory of Open Access Journals (Sweden)

Ricardo Arcanjo de Lima

2008-08-01

Full Text Available Este artigo visa fornecer uma visão global da atividade científica ibero-americana (América Latina + Espanha e Portugal em bioprospecção, através de indicadores bibliométricos unidimensionais a partir de documentos científicos indexados na base de dados multidisciplinar Science Citation Index (SCI. Por se tratar de uma área de pesquisa interdisciplinar, a bioprospecção é um objeto de estudo particularmente interessante para análise bibliométrica e pode ilustrar procedimentos metodológicos inovadores. Os resultados obtidos indicam que a despeito da existência de 7 países megadiversos nesta região geopolítica, a atividade científica em bioprospecção se encontra bastante concentrada no Brasil e na Espanha, refletindo, de certo modo, a fragilidade acadêmica dos outros países da região. Isso indica a necessidade de discutir e formular políticas claras, eficientes e sustentáveis dirigidas à bioprospecção, incluindo a administração da biodiversidade. Abstract This article provides a picture of scientific activity in bioprospecting in the Ibero-American geo-political region (Latin America + Spain + Portugal, on the basis of information collected in the Science Citation Index database. Bioprospecting is a particularly interesting field for bibliometric analysis because it is interdisciplinary and requires the development of innovative methodological procedures. The findings reveal that despite the presence of 7 mega-diverse countries in the region, Brazil and Spain concentrate most of the scientific production. This probably indicates the academic weakness in bioprospecting of the other Latin American countries. The results point out to the need of the countries in the region to discuss and design policy instruments to foster local capabilities in research of bioprospecting activities, including the management of their biodiversity resources.

Antibiotic Resistome: Improving Detection and Quantification Accuracy for Comparative Metagenomics.

Science.gov (United States)

Elbehery, Ali H A; Aziz, Ramy K; Siam, Rania

2016-04-01

The unprecedented rise of life-threatening antibiotic resistance (AR), combined with the unparalleled advances in DNA sequencing of genomes and metagenomes, has pushed the need for in silico detection of the resistance potential of clinical and environmental metagenomic samples through the quantification of AR genes (i.e., genes conferring antibiotic resistance). Therefore, determining an optimal methodology to quantitatively and accurately assess AR genes in a given environment is pivotal. Here, we optimized and improved existing AR detection methodologies from metagenomic datasets to properly consider AR-generating mutations in antibiotic target genes. Through comparative metagenomic analysis of previously published AR gene abundance in three publicly available metagenomes, we illustrate how mutation-generated resistance genes are either falsely assigned or neglected, which alters the detection and quantitation of the antibiotic resistome. In addition, we inspected factors influencing the outcome of AR gene quantification using metagenome simulation experiments, and identified that genome size, AR gene length, total number of metagenomics reads and selected sequencing platforms had pronounced effects on the level of detected AR. In conclusion, our proposed improvements in the current methodologies for accurate AR detection and resistome assessment show reliable results when tested on real and simulated metagenomic datasets.

Back to the Future of Soil Metagenomics.\

Czech Academy of Sciences Publication Activity Database

Nesme J, J.; Achouak, W.; Agathos SN, S.N.; Bailey, M.; Baldrian, Petr; Brunel, D.; Frostegård, Å.; Heulin, T.; Jansson JK, J.K.; Jurkevitch, E.; Kruus, K.L.; Kowalchuk, G.A.; Lagares, A.; Lapin-Scott, H.M.; Lemanceau, P.; Le Paslier, D.; Mandic-Mulec, I.; Murrell, J.C.; Myrold, D.D.; Nalin, R.; Nannipieri, P.; Neufeld, J.D.; O'Gara, F.; Parnell, J.J.; Pühler, A.; Pylro, V.; Ramos, J.L.; Roesch, L.F.; Schloter, M.; Schleper, C.; Sczyrba, A.; Sessitsch, A.; Sjöling, S.; Sørensen, J.; Sørensen, S.J.; Tebbe, C.C.; Topp, E.; Tsiamis, G.; van Elsas, J.D.; van Keulen, G.; Widmer, F.; Wagner, M.; Zhang, T.; Zhang, X.; Zhao, L; Zhu, Y-G.; Vogel, T.M.; Simonet, P.

2016-01-01

Roč. 7, FEB 10 (2016), s. 73 ISSN 1664-302X Institutional support: RVO:61388971 Keywords : metagenomic * soil microbiology; terrestrial microbiology * metagenomic; soil microbiology; terrestrial microbiology Subject RIV: EE - Microbiology, Virology Impact factor: 4.076, year: 2016

Metagenomics as a preliminary screen for antimicrobial bioprospecting

KAUST Repository

Al Amoudi, Soha; Razali, Rozaimi; Essack, Magbubah; Amini, Mohammad Shoaib; Bougouffa, Salim; Archer, John A.C.; Lafi, Feras Fawzi; Bajic, Vladimir B.

2016-01-01

DNA associated with antibiotic resistance. Moreover, our findings show an identical shift in phyla associated with historic hydrocarbon contamination exposure reported in previous studies (that is, enrichment of Gamma-and Delta-proteobacteria), but we

N-glycosylation in sugarcane

Directory of Open Access Journals (Sweden)

Maia Ivan G.

2001-01-01

Full Text Available The N-linked glycosylation of secretory and membrane proteins is the most complex posttranslational modification known to occur in eukaryotic cells. It has been shown to play critical roles in modulating protein function. Although this important biological process has been extensively studied in mammals, much less is known about this biosynthetic pathway in plants. The enzymes involved in plant N-glycan biosynthesis and processing are still not well defined and the mechanism of their genetic regulation is almost completely unknown. In this paper we describe our first attempt to understand the N-linked glycosylation mechanism in a plant species by using the data generated by the Sugarcane Expressed Sequence Tag (SUCEST project. The SUCEST database was mined for sugarcane gene products potentially involved in the N-glycosylation pathway. This approach has led to the identification and functional assignment of 90 expressed sequence tag (EST clusters sharing significant sequence similarity with the enzymes involved in N-glycan biosynthesis and processing. The ESTs identified were also analyzed to establish their relative abundance.

Analysis and comparison of very large metagenomes with fast clustering and functional annotation

Directory of Open Access Journals (Sweden)

Li Weizhong

2009-10-01

Full Text Available Abstract Background The remarkable advance of metagenomics presents significant new challenges in data analysis. Metagenomic datasets (metagenomes are large collections of sequencing reads from anonymous species within particular environments. Computational analyses for very large metagenomes are extremely time-consuming, and there are often many novel sequences in these metagenomes that are not fully utilized. The number of available metagenomes is rapidly increasing, so fast and efficient metagenome comparison methods are in great demand. Results The new metagenomic data analysis method Rapid Analysis of Multiple Metagenomes with a Clustering and Annotation Pipeline (RAMMCAP was developed using an ultra-fast sequence clustering algorithm, fast protein family annotation tools, and a novel statistical metagenome comparison method that employs a unique graphic interface. RAMMCAP processes extremely large datasets with only moderate computational effort. It identifies raw read clusters and protein clusters that may include novel gene families, and compares metagenomes using clusters or functional annotations calculated by RAMMCAP. In this study, RAMMCAP was applied to the two largest available metagenomic collections, the "Global Ocean Sampling" and the "Metagenomic Profiling of Nine Biomes". Conclusion RAMMCAP is a very fast method that can cluster and annotate one million metagenomic reads in only hundreds of CPU hours. It is available from http://tools.camera.calit2.net/camera/rammcap/.

Multiple comparative metagenomics using multiset k-mer counting

Directory of Open Access Journals (Sweden)

Gaëtan Benoit

2016-11-01

Full Text Available Background Large scale metagenomic projects aim to extract biodiversity knowledge between different environmental conditions. Current methods for comparing microbial communities face important limitations. Those based on taxonomical or functional assignation rely on a small subset of the sequences that can be associated to known organisms. On the other hand, de novo methods, that compare the whole sets of sequences, either do not scale up on ambitious metagenomic projects or do not provide precise and exhaustive results. Methods These limitations motivated the development of a new de novo metagenomic comparative method, called Simka. This method computes a large collection of standard ecological distances by replacing species counts by k-mer counts. Simka scales-up today’s metagenomic projects thanks to a new parallel k-mer counting strategy on multiple datasets. Results Experiments on public Human Microbiome Project datasets demonstrate that Simka captures the essential underlying biological structure. Simka was able to compute in a few hours both qualitative and quantitative ecological distances on hundreds of metagenomic samples (690 samples, 32 billions of reads. We also demonstrate that analyzing metagenomes at the k-mer level is highly correlated with extremely precise de novo comparison techniques which rely on all-versus-all sequences alignment strategy or which are based on taxonomic profiling.

Nutritional Therapies in Congenital Disorders of Glycosylation (CDG

Directory of Open Access Journals (Sweden)

Peter Witters

2017-11-01

Full Text Available Congenital disorders of glycosylation (CDG are a group of more than 130 inborn errors of metabolism affecting N-linked, O-linked protein and lipid-linked glycosylation. The phenotype in CDG patients includes frequent liver involvement, especially the disorders belonging to the N-linked protein glycosylation group. There are only a few treatable CDG. Mannose-Phosphate Isomerase (MPI-CDG was the first treatable CDG by high dose mannose supplements. Recently, with the successful use of d-galactose in Phosphoglucomutase 1 (PGM1-CDG, other CDG types have been trialed on galactose and with an increasing number of potential nutritional therapies. Current mini review focuses on therapies in glycosylation disorders affecting liver function and dietary intervention in general in N-linked glycosylation disorders. We also emphasize now the importance of early screening for CDG in patients with mild hepatopathy but also in cholestasis.

Genome signature analysis of thermal virus metagenomes reveals Archaea and thermophilic signatures.

Science.gov (United States)

Pride, David T; Schoenfeld, Thomas

2008-09-17

Metagenomic analysis provides a rich source of biological information for otherwise intractable viral communities. However, study of viral metagenomes has been hampered by its nearly complete reliance on BLAST algorithms for identification of DNA sequences. We sought to develop algorithms for examination of viral metagenomes to identify the origin of sequences independent of BLAST algorithms. We chose viral metagenomes obtained from two hot springs, Bear Paw and Octopus, in Yellowstone National Park, as they represent simple microbial populations where comparatively large contigs were obtained. Thermal spring metagenomes have high proportions of sequences without significant Genbank homology, which has hampered identification of viruses and their linkage with hosts. To analyze each metagenome, we developed a method to classify DNA fragments using genome signature-based phylogenetic classification (GSPC), where metagenomic fragments are compared to a database of oligonucleotide signatures for all previously sequenced Bacteria, Archaea, and viruses. From both Bear Paw and Octopus hot springs, each assembled contig had more similarity to other metagenome contigs than to any sequenced microbial genome based on GSPC analysis, suggesting a genome signature common to each of these extreme environments. While viral metagenomes from Bear Paw and Octopus share some similarity, the genome signatures from each locale are largely unique. GSPC using a microbial database predicts most of the Octopus metagenome has archaeal signatures, while bacterial signatures predominate in Bear Paw; a finding consistent with those of Genbank BLAST. When using a viral database, the majority of the Octopus metagenome is predicted to belong to archaeal virus Families Globuloviridae and Fuselloviridae, while none of the Bear Paw metagenome is predicted to belong to archaeal viruses. As expected, when microbial and viral databases are combined, each of the Octopus and Bear Paw metagenomic contigs

Unsupervised Two-Way Clustering of Metagenomic Sequences

Directory of Open Access Journals (Sweden)

Shruthi Prabhakara

2012-01-01

Full Text Available A major challenge facing metagenomics is the development of tools for the characterization of functional and taxonomic content of vast amounts of short metagenome reads. The efficacy of clustering methods depends on the number of reads in the dataset, the read length and relative abundances of source genomes in the microbial community. In this paper, we formulate an unsupervised naive Bayes multispecies, multidimensional mixture model for reads from a metagenome. We use the proposed model to cluster metagenomic reads by their species of origin and to characterize the abundance of each species. We model the distribution of word counts along a genome as a Gaussian for shorter, frequent words and as a Poisson for longer words that are rare. We employ either a mixture of Gaussians or mixture of Poissons to model reads within each bin. Further, we handle the high-dimensionality and sparsity associated with the data, by grouping the set of words comprising the reads, resulting in a two-way mixture model. Finally, we demonstrate the accuracy and applicability of this method on simulated and real metagenomes. Our method can accurately cluster reads as short as 100 bps and is robust to varying abundances, divergences and read lengths.

Critical Assessment of Metagenome Interpretation-a benchmark of metagenomics software.

Science.gov (United States)

Sczyrba, Alexander; Hofmann, Peter; Belmann, Peter; Koslicki, David; Janssen, Stefan; Dröge, Johannes; Gregor, Ivan; Majda, Stephan; Fiedler, Jessika; Dahms, Eik; Bremges, Andreas; Fritz, Adrian; Garrido-Oter, Ruben; Jørgensen, Tue Sparholt; Shapiro, Nicole; Blood, Philip D; Gurevich, Alexey; Bai, Yang; Turaev, Dmitrij; DeMaere, Matthew Z; Chikhi, Rayan; Nagarajan, Niranjan; Quince, Christopher; Meyer, Fernando; Balvočiūtė, Monika; Hansen, Lars Hestbjerg; Sørensen, Søren J; Chia, Burton K H; Denis, Bertrand; Froula, Jeff L; Wang, Zhong; Egan, Robert; Don Kang, Dongwan; Cook, Jeffrey J; Deltel, Charles; Beckstette, Michael; Lemaitre, Claire; Peterlongo, Pierre; Rizk, Guillaume; Lavenier, Dominique; Wu, Yu-Wei; Singer, Steven W; Jain, Chirag; Strous, Marc; Klingenberg, Heiner; Meinicke, Peter; Barton, Michael D; Lingner, Thomas; Lin, Hsin-Hung; Liao, Yu-Chieh; Silva, Genivaldo Gueiros Z; Cuevas, Daniel A; Edwards, Robert A; Saha, Surya; Piro, Vitor C; Renard, Bernhard Y; Pop, Mihai; Klenk, Hans-Peter; Göker, Markus; Kyrpides, Nikos C; Woyke, Tanja; Vorholt, Julia A; Schulze-Lefert, Paul; Rubin, Edward M; Darling, Aaron E; Rattei, Thomas; McHardy, Alice C

2017-11-01

Methods for assembly, taxonomic profiling and binning are key to interpreting metagenome data, but a lack of consensus about benchmarking complicates performance assessment. The Critical Assessment of Metagenome Interpretation (CAMI) challenge has engaged the global developer community to benchmark their programs on highly complex and realistic data sets, generated from ∼700 newly sequenced microorganisms and ∼600 novel viruses and plasmids and representing common experimental setups. Assembly and genome binning programs performed well for species represented by individual genomes but were substantially affected by the presence of related strains. Taxonomic profiling and binning programs were proficient at high taxonomic ranks, with a notable performance decrease below family level. Parameter settings markedly affected performance, underscoring their importance for program reproducibility. The CAMI results highlight current challenges but also provide a roadmap for software selection to answer specific research questions.

Structural and mechanistic analysis of a β-glycoside phosphorylase identified by screening a metagenomic library.

Science.gov (United States)

Macdonald, Spencer S; Patel, Ankoor; Larmour, Veronica L C; Morgan-Lang, Connor; Hallam, Steven J; Mark, Brian L; Withers, Stephen G

2018-03-02

Glycoside phosphorylases have considerable potential as catalysts for the assembly of useful glycans for products ranging from functional foods and prebiotics to novel materials. However, the substrate diversity of currently identified phosphorylases is relatively small, limiting their practical applications. To address this limitation, we developed a high-throughput screening approach using the activated substrate 2,4-dinitrophenyl β-d-glucoside (DNPGlc) and inorganic phosphate for identifying glycoside phosphorylase activity and used it to screen a large insert metagenomic library. The initial screen, based on release of 2,4-dinitrophenyl from DNPGlc in the presence of phosphate, identified the gene bglP, encoding a retaining β-glycoside phosphorylase from the CAZy GH3 family. Kinetic and mechanistic analysis of the gene product, BglP, confirmed a double displacement ping-pong mechanism involving a covalent glycosyl-enzyme intermediate. X-ray crystallographic analysis provided insights into the phosphate-binding mode and identified a key glutamine residue in the active site important for substrate recognition. Substituting this glutamine for a serine swapped the substrate specificity from glucoside to N -acetylglucosaminide. In summary, we present a high-throughput screening approach for identifying β-glycoside phosphorylases, which was robust, simple to implement, and useful in identifying active clones within a metagenomics library. Implementation of this screen enabled discovery of a new glycoside phosphorylase class and has paved the way to devising simple ways in which enzyme specificity can be encoded and swapped, which has implications for biotechnological applications. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Genome signature analysis of thermal virus metagenomes reveals Archaea and thermophilic signatures

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Pride David T

2008-09-01

Full Text Available Abstract Background Metagenomic analysis provides a rich source of biological information for otherwise intractable viral communities. However, study of viral metagenomes has been hampered by its nearly complete reliance on BLAST algorithms for identification of DNA sequences. We sought to develop algorithms for examination of viral metagenomes to identify the origin of sequences independent of BLAST algorithms. We chose viral metagenomes obtained from two hot springs, Bear Paw and Octopus, in Yellowstone National Park, as they represent simple microbial populations where comparatively large contigs were obtained. Thermal spring metagenomes have high proportions of sequences without significant Genbank homology, which has hampered identification of viruses and their linkage with hosts. To analyze each metagenome, we developed a method to classify DNA fragments using genome signature-based phylogenetic classification (GSPC, where metagenomic fragments are compared to a database of oligonucleotide signatures for all previously sequenced Bacteria, Archaea, and viruses. Results From both Bear Paw and Octopus hot springs, each assembled contig had more similarity to other metagenome contigs than to any sequenced microbial genome based on GSPC analysis, suggesting a genome signature common to each of these extreme environments. While viral metagenomes from Bear Paw and Octopus share some similarity, the genome signatures from each locale are largely unique. GSPC using a microbial database predicts most of the Octopus metagenome has archaeal signatures, while bacterial signatures predominate in Bear Paw; a finding consistent with those of Genbank BLAST. When using a viral database, the majority of the Octopus metagenome is predicted to belong to archaeal virus Families Globuloviridae and Fuselloviridae, while none of the Bear Paw metagenome is predicted to belong to archaeal viruses. As expected, when microbial and viral databases are combined, each of

Halide-mediated regioselective 6-O-glycosylation of unprotected hexopyranosides with perbenzylated glycosyl bromide donors

DEFF Research Database (Denmark)

Niedbal, Dominika Alina; Madsen, Robert

2016-01-01

The regio- and stereoselective glycosylation at the 6-position in 2,3,4,6-unprotected hexopyranosides has been investigated with dibutyltin oxide as the directing agent. Perbenzylated hexopyranosyl bromides were employed as the donors and the glycosylations were promoted by tetrabutylammonium...... bromide. The couplings were completely selective for both glucose and galactose donors and acceptors as long as the stannylene acetal of the acceptor was soluble in dichloromethane. This gave rise to a number of 1,2-cis-linked disaccharides in reasonable yields. Mannose donors and acceptors, on the other...

Glycosylation of the self-recognizing Escherichia coli Ag43 autotransporter protein

DEFF Research Database (Denmark)

Sherlock, O.; Dobrindt, U.; Jensen, J.B.

2006-01-01

a novel member to this exclusive group, namely, antigen 43 (Ag43), a self-recognizing autotransporter protein. By mass spectrometry Ag43 was demonstrated to be glycosylated by addition of heptose residues at several positions in the passenger domain. Glycosylation of Ag43 by the action of the Aah and Tib......C glycosyltransferases was observed in laboratory strains. Importantly, Ag43 was also found to be glycosylated in a wild-type strain, suggesting that Ag43-glycosylation may be a widespread phenomenon. Glycosylation of Ag43 does not seem to interfere with its self-associating properties. However, the glycosylated form...

Digestibility and IgE-Binding of Glycosylated Codfish Parvalbumin

Science.gov (United States)

de Jongh, Harmen H. J.; Robles, Carlos López; Nordlee, Julie A.; Lee, Poi-Wah; Baumert, Joseph L.; Hamilton, Robert G.; Taylor, Steve L.; Koppelman, Stef J.

2013-01-01

Food-processing conditions may alter the allergenicity of food proteins by different means. In this study, the effect of the glycosylation as a result of thermal treatment on the digestibility and IgE-binding of codfish parvalbumin is investigated. Native and glycosylated parvalbumins were digested with pepsin at various conditions relevant for the gastrointestinal tract. Intact proteins and peptides were analysed for apparent molecular weight and IgE-binding. Glycosylation did not substantially affect the digestion. Although the peptides resulting from digestion were relatively large (3 and 4 kDa), the IgE-binding was strongly diminished. However, the glycosylated parvalbumin had a strong propensity to form dimers and tetramers, and these multimers bound IgE intensely, suggesting stronger IgE-binding than monomeric parvalbumin. We conclude that glycosylation of codfish parvalbumin does not affect the digestibility of parvalbumin and that the peptides resulting from this digestion show low IgE-binding, regardless of glycosylation. Glycosylation of parvalbumin leads to the formation of higher order structures that are more potent IgE binders than native, monomeric parvalbumin. Therefore, food-processing conditions applied to fish allergen can potentially lead to increased allergenicity, even while the protein's digestibility is not affected by such processing. PMID:23878817

Digestibility and IgE-Binding of Glycosylated Codfish Parvalbumin

Directory of Open Access Journals (Sweden)

Harmen H. J. de Jongh

2013-01-01

Full Text Available Food-processing conditions may alter the allergenicity of food proteins by different means. In this study, the effect of the glycosylation as a result of thermal treatment on the digestibility and IgE-binding of codfish parvalbumin is investigated. Native and glycosylated parvalbumins were digested with pepsin at various conditions relevant for the gastrointestinal tract. Intact proteins and peptides were analysed for apparent molecular weight and IgE-binding. Glycosylation did not substantially affect the digestion. Although the peptides resulting from digestion were relatively large (3 and 4 kDa, the IgE-binding was strongly diminished. However, the glycosylated parvalbumin had a strong propensity to form dimers and tetramers, and these multimers bound IgE intensely, suggesting stronger IgE-binding than monomeric parvalbumin. We conclude that glycosylation of codfish parvalbumin does not affect the digestibility of parvalbumin and that the peptides resulting from this digestion show low IgE-binding, regardless of glycosylation. Glycosylation of parvalbumin leads to the formation of higher order structures that are more potent IgE binders than native, monomeric parvalbumin. Therefore, food-processing conditions applied to fish allergen can potentially lead to increased allergenicity, even while the protein’s digestibility is not affected by such processing.

Functional Analysis of Glycosylation of Zika Virus Envelope Protein

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Camila R. Fontes-Garfias

2017-10-01

Full Text Available Summary: Zika virus (ZIKV infection causes devastating congenital abnormities and Guillain-Barré syndrome. The ZIKV envelope (E protein is responsible for viral entry and represents a major determinant for viral pathogenesis. Like other flaviviruses, the ZIKV E protein is glycosylated at amino acid N154. To study the function of E glycosylation, we generated a recombinant N154Q ZIKV that lacks the E glycosylation and analyzed the mutant virus in mammalian and mosquito hosts. In mouse models, the mutant was attenuated, as evidenced by lower viremia, decreased weight loss, and no mortality; however, knockout of E glycosylation did not significantly affect neurovirulence. Mice immunized with the mutant virus developed a robust neutralizing antibody response and were completely protected from wild-type ZIKV challenge. In mosquitoes, the mutant virus exhibited diminished oral infectivity for the Aedes aegypti vector. Collectively, the results demonstrate that E glycosylation is critical for ZIKV infection of mammalian and mosquito hosts. : Zika virus (ZIKV causes devastating congenital abnormities and Guillain-Barré syndrome. Fontes-Garfias et al. showed that the glycosylation of ZIKV envelope protein plays an important role in infecting mosquito vectors and pathogenesis in mouse. Keywords: Zika virus, glycosylation, flavivirus entry, mosquito transmission, vaccine

Toward stable genetic engineering of human o-glycosylation in plants

DEFF Research Database (Denmark)

Yang, Zhang; Bennett, Eric Paul; Jørgensen, Bodil

2012-01-01

Glycosylation is the most abundant and complex posttranslational modification to be considered for recombinant production of therapeutic proteins. Mucin-type (N-acetylgalactosamine [GalNAc]-type) O-glycosylation is found in eumetazoan cells but absent in plants and yeast, making these cell types...... an obvious choice for de novo engineering of this O-glycosylation pathway. We previously showed that transient implementation of O-glycosylation capacity in plants requires introduction of the synthesis of the donor substrate UDP-GalNAc and one or more polypeptide GalNAc-transferases for incorporating Gal......NAc residues into proteins. Here, we have stably engineered O-glycosylation capacity in two plant cell systems, soil-grown Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) Bright Yellow-2 suspension culture cells. Efficient GalNAc O-glycosylation of two stably coexpressed substrate O...

Glucose-tolerant β-glucosidase retrieved from the metagenome

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Taku eUchiyama

2015-06-01

Full Text Available β-glucosidases (BGLs hydrolyze cellooligosaccharides to glucose and play a crucial role in the enzymatic saccharification of cellulosic biomass. Despite their significance for the production of glucose, most identified BGLs are commonly inhibited by low (~mM concentrations of glucose. Therefore, BGLs that are insensitive to glucose inhibition have great biotechnological merit. We applied a metagenomic approach to screen for such rare glucose-tolerant BGLs. A metagenomic library was created in Escherichia coli (approximately 10,000 colonies and grown on LB agar plates containing 5-bromo-4-chloro-3-indolyl-β-D-glucoside, yielding 828 positive (blue colonies. These were then arrayed in 96-well plates, grown in LB, and secondarily screened for activity in the presence of 10% (w/v glucose. Seven glucose-tolerant clones were identified, each of which contained a single bgl gene. The genes were classified into two groups, differing by two nucleotides. The deduced amino acid sequences of these genes were identical (452 aa and found to belong to the glycosyl hydrolase family 1. The recombinant protein (Ks5A7 was overproduced in E. coli as a C-terminal 6 × His-tagged protein and purified to apparent homogeneity. The molecular mass of the purified Ks5A7 was determined to be 54 kDa by SDS-PAGE, and 160 kDa by gel filtration analysis. The enzyme was optimally active at 45°C and pH 5.0–6.5 and retained full or 1.5–2-fold enhanced activity in the presence of 0.1–0.5 M glucose. It had a low KM (78 µM with p-nitrophenyl β-D-glucoside; 0.36 mM with cellobiose and high Vmax (91 µmol min-1 mg-1 with p-nitrophenyl β-D-glucoside; 155 µmol min-1 mg-1 with cellobiose among known glucose-tolerant BGLs and was free from substrate (0.1 M cellobiose inhibition. The efficient use of Ks5A7 in conjunction with Trichoderma reesei cellulases in enzymatic saccharification of alkaline-treated rice straw was demonstrated by increased production of glucose.

Trans-species Engineering of Glycosylated Therapeutic Proteins

DEFF Research Database (Denmark)

Yang, Zhang

eukaryotes and even prokaryotes. Insect and yeast cells produce O-glycosylation incompatible with use in humans, however recently the yeast Pichia was engineered to perform the first step of human-like O-glycosylation. This review provides an overview of past and current engineering efforts of N...

Exploiting HPC Platforms for Metagenomics: Challenges and Opportunities (MICW - Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

Energy Technology Data Exchange (ETDEWEB)

Canon, Shane

2011-10-12

DOE JGI's Zhong Wang, chair of the High-performance Computing session, gives a brief introduction before Berkeley Lab's Shane Canon talks about "Exploiting HPC Platforms for Metagenomics: Challenges and Opportunities" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

Marine Metagenome as A Resource for Novel Enzymes

KAUST Repository

Alma’ abadi, Amani D.; Gojobori, Takashi; Mineta, Katsuhiko

2015-01-01

the metagenomics approach has many limitations, it is expected to provide not only scientific insights but also economic benefits, especially in industry. This review highlights the importance of metagenomics in mining microbial lipases, as an example, by using

Similarities and Differences in the Glycosylation Mechanisms in Prokaryotes and Eukaryotes

Directory of Open Access Journals (Sweden)

Anne Dell

2010-01-01

Full Text Available Recent years have witnessed a rapid growth in the number and diversity of prokaryotic proteins shown to carry N- and/or O-glycans, with protein glycosylation now considered as fundamental to the biology of these organisms as it is in eukaryotic systems. This article overviews the major glycosylation pathways that are known to exist in eukarya, bacteria and archaea. These are (i oligosaccharyltransferase (OST-mediated N-glycosylation which is abundant in eukarya and archaea, but is restricted to a limited range of bacteria; (ii stepwise cytoplasmic N-glycosylation that has so far only been confirmed in the bacterial domain; (iii OST-mediated O-glycosylation which appears to be characteristic of bacteria; and (iv stepwise O-glycosylation which is common in eukarya and bacteria. A key aim of the review is to integrate information from the three domains of life in order to highlight commonalities in glycosylation processes. We show how the OST-mediated N- and O-glycosylation pathways share cytoplasmic assembly of lipid-linked oligosaccharides, flipping across the ER/periplasmic/cytoplasmic membranes, and transferring “en bloc” to the protein acceptor. Moreover these hallmarks are mirrored in lipopolysaccharide biosynthesis. Like in eukaryotes, stepwise O-glycosylation occurs on diverse bacterial proteins including flagellins, adhesins, autotransporters and lipoproteins, with O-glycosylation chain extension often coupled with secretory mechanisms.

Metagenomic Sequencing of an In Vitro-Simulated Microbial Community

Energy Technology Data Exchange (ETDEWEB)

Morgan, Jenna L.; Darling, Aaron E.; Eisen, Jonathan A.

2009-12-01

Background: Microbial life dominates the earth, but many species are difficult or even impossible to study under laboratory conditions. Sequencing DNA directly from the environment, a technique commonly referred to as metagenomics, is an important tool for cataloging microbial life. This culture-independent approach involves collecting samples that include microbes in them, extracting DNA from the samples, and sequencing the DNA. A sample may contain many different microorganisms, macroorganisms, and even free-floating environmental DNA. A fundamental challenge in metagenomics has been estimating the abundance of organisms in a sample based on the frequency with which the organism's DNA was observed in reads generated via DNA sequencing. Methodology/Principal Findings: We created mixtures of ten microbial species for which genome sequences are known. Each mixture contained an equal number of cells of each species. We then extracted DNA from the mixtures, sequenced the DNA, and measured the frequency with which genomic regions from each organism was observed in the sequenced DNA. We found that the observed frequency of reads mapping to each organism did not reflect the equal numbers of cells that were known to be included in each mixture. The relative organism abundances varied significantly depending on the DNA extraction and sequencing protocol utilized. Conclusions/Significance: We describe a new data resource for measuring the accuracy of metagenomic binning methods, created by in vitro-simulation of a metagenomic community. Our in vitro simulation can be used to complement previous in silico benchmark studies. In constructing a synthetic community and sequencing its metagenome, we encountered several sources of observation bias that likely affect most metagenomic experiments to date and present challenges for comparative metagenomic studies. DNA preparation methods have a particularly profound effect in our study, implying that samples prepared with

Glycosylation of the N-terminal potential N-glycosylation sites in the human α1,3-fucosyltransferase V and -VI (hFucTV and -VI)

DEFF Research Database (Denmark)

Christensen, Lise Lotte; Bross, Peter Gerd; Ørntoft, Torben Falck

2000-01-01

Human alpha1,3-fucosyltransferase V and -VI (hFucTV and -VI) each contain four potential N-glycosylation sites (hFucTV: Asn60, Asn105, Asn167 and Asn198 and hFucTVI: Asn46, Asn91, Asn153 and Asn184). Glycosylation of the two N-terminal potential N-glycosylation sites (hFucTV: Asn60, Asn105 and h......FucTVI: Asn46 and Asn91) have never been studied in detail. In the present study, we have analysed the glycosylation of these potential N-glycosylation sites. Initially, we compared the molecular mass of hFucTV and -VI expressed in COS-7 cells treated with tunicamycin with the mass of the proteins...... in untreated cells. The difference in molecular mass between the proteins in treated and untreated cells corresponded to the presence of at least three N-linked glycans. We then made a series of mutants, in which the asparagine residues in the N-terminal potential N-glycosylation sites were replaced...

Diversity and functions of protein glycosylation in insects.

Science.gov (United States)

Walski, Tomasz; De Schutter, Kristof; Van Damme, Els J M; Smagghe, Guy

2017-04-01

The majority of proteins is modified with carbohydrate structures. This modification, called glycosylation, was shown to be crucial for protein folding, stability and subcellular location, as well as protein-protein interactions, recognition and signaling. Protein glycosylation is involved in multiple physiological processes, including embryonic development, growth, circadian rhythms, cell attachment as well as maintenance of organ structure, immunity and fertility. Although the general principles of glycosylation are similar among eukaryotic organisms, insects synthesize a distinct repertoire of glycan structures compared to plants and vertebrates. Consequently, a number of unique insect glycans mediate functions specific to this class of invertebrates. For instance, the core α1,3-fucosylation of N-glycans is absent in vertebrates, while in insects this modification is crucial for the development of wings and the nervous system. At present, most of the data on insect glycobiology comes from research in Drosophila. Yet, progressively more information on the glycan structures and the importance of glycosylation in other insects like beetles, caterpillars, aphids and bees is becoming available. This review gives a summary of the current knowledge and recent progress related to glycan diversity and function(s) of protein glycosylation in insects. We focus on N- and O-glycosylation, their synthesis, physiological role(s), as well as the molecular and biochemical basis of these processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Assembly of viral genomes from metagenomes

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Saskia L Smits

2014-12-01

Full Text Available Viral infections remain a serious global health issue. Metagenomic approaches are increasingly used in the detection of novel viral pathogens but also to generate complete genomes of uncultivated viruses. In silico identification of complete viral genomes from sequence data would allow rapid phylogenetic characterization of these new viruses. Often, however, complete viral genomes are not recovered, but rather several distinct contigs derived from a single entity, some of which have no sequence homology to any known proteins. De novo assembly of single viruses from a metagenome is challenging, not only because of the lack of a reference genome, but also because of intrapopulation variation and uneven or insufficient coverage. Here we explored different assembly algorithms, remote homology searches, genome-specific sequence motifs, k-mer frequency ranking, and coverage profile binning to detect and obtain viral target genomes from metagenomes. All methods were tested on 454-generated sequencing datasets containing three recently described RNA viruses with a relatively large genome which were divergent to previously known viruses from the viral families Rhabdoviridae and Coronaviridae. Depending on specific characteristics of the target virus and the metagenomic community, different assembly and in silico gap closure strategies were successful in obtaining near complete viral genomes.

BioCreative Workshops for DOE Genome Sciences: Text Mining for Metagenomics

Energy Technology Data Exchange (ETDEWEB)

Wu, Cathy H. [Univ. of Delaware, Newark, DE (United States). Center for Bioinformatics and Computational Biology; Hirschman, Lynette [The MITRE Corporation, Bedford, MA (United States)

2016-10-29

The objective of this project was to host BioCreative workshops to define and develop text mining tasks to meet the needs of the Genome Sciences community, focusing on metadata information extraction in metagenomics. Following the successful introduction of metagenomics at the BioCreative IV workshop, members of the metagenomics community and BioCreative communities continued discussion to identify candidate topics for a BioCreative metagenomics track for BioCreative V. Of particular interest was the capture of environmental and isolation source information from text. The outcome was to form a “community of interest” around work on the interactive EXTRACT system, which supported interactive tagging of environmental and species data. This experiment is included in the BioCreative V virtual issue of Database. In addition, there was broad participation by members of the metagenomics community in the panels held at BioCreative V, leading to valuable exchanges between the text mining developers and members of the metagenomics research community. These exchanges are reflected in a number of the overview and perspective pieces also being captured in the BioCreative V virtual issue. Overall, this conversation has exposed the metagenomics researchers to the possibilities of text mining, and educated the text mining developers to the specific needs of the metagenomics community.

Evaluation of ddRADseq for reduced representation metagenome sequencing

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Michael Y. Liu

2017-09-01

Full Text Available Background Profiling of microbial communities via metagenomic shotgun sequencing has enabled researches to gain unprecedented insight into microbial community structure and the functional roles of community members. This study describes a method and basic analysis for a metagenomic adaptation of the double digest restriction site associated DNA sequencing (ddRADseq protocol for reduced representation metagenome profiling. Methods This technique takes advantage of the sequence specificity of restriction endonucleases to construct an Illumina-compatible sequencing library containing DNA fragments that are between a pair of restriction sites located within close proximity. This results in a reduced sequencing library with coverage breadth that can be tuned by size selection. We assessed the performance of the metagenomic ddRADseq approach by applying the full method to human stool samples and generating sequence data. Results The ddRADseq data yields a similar estimate of community taxonomic profile as obtained from shotgun metagenome sequencing of the same human stool samples. No obvious bias with respect to genomic G + C content and the estimated relative species abundance was detected. Discussion Although ddRADseq does introduce some bias in taxonomic representation, the bias is likely to be small relative to DNA extraction bias. ddRADseq appears feasible and could have value as a tool for metagenome-wide association studies.

Tentacle: distributed quantification of genes in metagenomes.

Science.gov (United States)

Boulund, Fredrik; Sjögren, Anders; Kristiansson, Erik

2015-01-01

In metagenomics, microbial communities are sequenced at increasingly high resolution, generating datasets with billions of DNA fragments. Novel methods that can efficiently process the growing volumes of sequence data are necessary for the accurate analysis and interpretation of existing and upcoming metagenomes. Here we present Tentacle, which is a novel framework that uses distributed computational resources for gene quantification in metagenomes. Tentacle is implemented using a dynamic master-worker approach in which DNA fragments are streamed via a network and processed in parallel on worker nodes. Tentacle is modular, extensible, and comes with support for six commonly used sequence aligners. It is easy to adapt Tentacle to different applications in metagenomics and easy to integrate into existing workflows. Evaluations show that Tentacle scales very well with increasing computing resources. We illustrate the versatility of Tentacle on three different use cases. Tentacle is written for Linux in Python 2.7 and is published as open source under the GNU General Public License (v3). Documentation, tutorials, installation instructions, and the source code are freely available online at: http://bioinformatics.math.chalmers.se/tentacle.

SmashCommunity: A metagenomic annotation and analysis tool

DEFF Research Database (Denmark)

Arumugam, Manimozhiyan; Harrington, Eoghan D; Foerstner, Konrad U

2010-01-01

the quantitative phylogenetic and functional compositions of metagenomes, to compare compositions of multiple metagenomes and to produce intuitive visual representations of such analyses. AVAILABILITY: SmashCommunity is freely available at http://www.bork.embl.de/software/smash CONTACT: bork@embl.de....

FANTOM: Functional and taxonomic analysis of metagenomes

Directory of Open Access Journals (Sweden)

Sanli Kemal

2013-02-01

Full Text Available Abstract Background Interpretation of quantitative metagenomics data is important for our understanding of ecosystem functioning and assessing differences between various environmental samples. There is a need for an easy to use tool to explore the often complex metagenomics data in taxonomic and functional context. Results Here we introduce FANTOM, a tool that allows for exploratory and comparative analysis of metagenomics abundance data integrated with metadata information and biological databases. Importantly, FANTOM can make use of any hierarchical database and it comes supplied with NCBI taxonomic hierarchies as well as KEGG Orthology, COG, PFAM and TIGRFAM databases. Conclusions The software is implemented in Python, is platform independent, and is available at http://www.sysbio.se/Fantom.

Constructing and screening a metagenomic library of a cold and alkaline extreme environment

DEFF Research Database (Denmark)

Glaring, Mikkel Andreas; Vester, Jan Kjølhede; Stougaard, Peter

2017-01-01

Natural cold or alkaline environments are common on Earth. A rare combination of these two extremes is found in the permanently cold (less than 6 °C) and alkaline (pH above 10) ikaite columns in the Ikka Fjord in Southern Greenland. Bioprospecting efforts have established the ikaite columns...

Towards diagnostic metagenomics of Campylobacter in fecal samples

DEFF Research Database (Denmark)

Andersen, Sandra Christine; Kiil, Kristoffer; Harder, Christoffer Bugge

2017-01-01

The development of diagnostic metagenomics is driven by the need for universal, culture-independent methods for detection and characterization of pathogens to substitute the time-consuming, organism-specific, and often culture-based laboratory procedures for epidemiological source-tracing. Some...... of the challenges in diagnostic metagenomics are, that it requires a great next-generation sequencing depth and unautomated data analysis. DNA from human fecal samples spiked with 7.75 × 101-7.75 × 107 colony forming unit (CFU)/ml Campylobacter jejuni and chicken fecal samples spiked with 1 × 102-1 × 106 CFU...... Campylobacter in all the clinical samples. Sensitivity in diagnostic metagenomics is improving and has reached a clinically relevant level. There are still challenges to overcome before real-time diagnostic metagenomics can replace quantitative polymerase chain reaction (qPCR) or culture-based surveillance...

Glycosylation in HIV-1 envelope glycoprotein and its biological implications

KAUST Repository

Ho, Yung Shwen

2013-08-01

Glycosylation of HIV-1 envelope proteins (Env gp120/gp41) plays a vital role in viral evasion from the host immune response, which occurs through the masking of key neutralization epitopes and the presentation of the Env glycosylation as \\'self\\' to the host immune system. Env glycosylation is generally conserved, yet its continual evolution plays an important role in modulating viral infectivity and Env immunogenicity. Thus, it is believed that Env glycosylation, which is a vital part of the HIV-1 architecture, also controls intra- and inter-clade genetic variations. Discerning intra- and inter-clade glycosylation variations could therefore yield important information for understanding the molecular and biological differences between HIV clades and may assist in effectively designing Env-based immunogens and in clearly understanding HIV vaccines. This review provides an in-depth perspective of various aspects of Env glycosylation in the context of HIV-1 pathogenesis. © 2013 Future Medicine Ltd.

[Non-enzymatic glycosylation of dietary protein in vitro].

Science.gov (United States)

Bednykh, B S; Evdokimov, I A; Sokolov, A I

2015-01-01

Non-enzymatic glycosylation of proteins, based on discovered by Mayarn reaction of carbohydrate aldehyde group with a free amino group of a protein molecule, is well known to experts in biochemistry of food industry. Generated brown solid in some cases give the product marketable qualities--crackling bread--in others conversely, worsen the product. The biological effects of far-advanced products of non-enzymatic protein glycosylation reaction have not been studied enough, although it was reported previously that they are not split by digestive enzymes and couldn't be absorbed by animals. The objective of this work was to compare the depth of glycosylation of different food proteins of animal and vegetable origin. The objects of the study were proteins of animal (casein, lactoglobulin, albumin) and vegetable (soy isolate, proteins of rice flour, buckwheat, oatmeal) origin, glucose and fructose were selected as glycosylation agents, exposure 15 days at 37 degrees C. Lactoglobulin was glycosylated to a lesser extent among the proteins of animal origin while protein of oatmeal was glycosylated in the least degree among vegetable proteins. Conversely, such proteins as casein and soya isolate protein bound rather large amounts of carbohydrates. Fructose binding with protein was generally higher than the binding of glucose. The only exception was a protein of oatmeal. When of glucose and fructose simultaneously presented in the incubation medium, glucose binding usually increased while binding of fructose, in contrast, reduced. According to the total amount of carbohydrate (mcg), which is able to attach a protein (mg) the studied food proteins located in the following order: albumin (38) > soy protein isolate (23) > casein (15,) > whey protein rice flour protein (6) > protein from buckwheat flour (3) > globulin (2) > protein of oatmeal (0.3). The results obtained are to be used to select the optimal combination of proteins and carbohydrates, in which the glycosylation

Effective Analysis of NGS Metagenomic Data with Ultra-Fast Clustering Algorithms (MICW - Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

Energy Technology Data Exchange (ETDEWEB)

Li, Weizhong

2011-10-12

San Diego Supercomputer Center's Weizhong Li on "Effective Analysis of NGS Metagenomic Data with Ultra-fast Clustering Algorithms" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

Challenges and Opportunities of Airborne Metagenomics

OpenAIRE

Behzad, Hayedeh; Gojobori, Takashi; Mineta, Katsuhiko

2015-01-01

Recent metagenomic studies of environments, such as marine and soil, have significantly enhanced our understanding of the diverse microbial communities living in these habitats and their essential roles in sustaining vast ecosystems. The increase in the number of publications related to soil and marine metagenomics is in sharp contrast to those of air, yet airborne microbes are thought to have significant impacts on many aspects of our lives from their potential roles in atmospheric events su...

Metagenomic analysis of permafrost microbial community response to thaw

Energy Technology Data Exchange (ETDEWEB)

Mackelprang, R.; Waldrop, M.P.; DeAngelis, K.M.; David, M.M.; Chavarria, K.L.; Blazewicz, S.J.; Rubin, E.M.; Jansson, J.K.

2011-07-01

We employed deep metagenomic sequencing to determine the impact of thaw on microbial phylogenetic and functional genes and related this data to measurements of methane emissions. Metagenomics, the direct sequencing of DNA from the environment, allows for the examination of whole biochemical pathways and associated processes, as opposed to individual pieces of the metabolic puzzle. Our metagenome analyses revealed that during transition from a frozen to a thawed state there were rapid shifts in many microbial, phylogenetic and functional gene abundances and pathways. After one week of incubation at 5°C, permafrost metagenomes converged to be more similar to each other than while they were frozen. We found that multiple genes involved in cycling of C and nitrogen shifted rapidly during thaw. We also constructed the first draft genome from a complex soil metagenome, which corresponded to a novel methanogen. Methane previously accumulated in permafrost was released during thaw and subsequently consumed by methanotrophic bacteria. Together these data point towards the importance of rapid cycling of methane and nitrogen in thawing permafrost.

Functional Analysis of Glycosylation of Zika Virus Envelope Protein.

Science.gov (United States)

Fontes-Garfias, Camila R; Shan, Chao; Luo, Huanle; Muruato, Antonio E; Medeiros, Daniele B A; Mays, Elizabeth; Xie, Xuping; Zou, Jing; Roundy, Christopher M; Wakamiya, Maki; Rossi, Shannan L; Wang, Tian; Weaver, Scott C; Shi, Pei-Yong

2017-10-31

Zika virus (ZIKV) infection causes devastating congenital abnormities and Guillain-Barré syndrome. The ZIKV envelope (E) protein is responsible for viral entry and represents a major determinant for viral pathogenesis. Like other flaviviruses, the ZIKV E protein is glycosylated at amino acid N154. To study the function of E glycosylation, we generated a recombinant N154Q ZIKV that lacks the E glycosylation and analyzed the mutant virus in mammalian and mosquito hosts. In mouse models, the mutant was attenuated, as evidenced by lower viremia, decreased weight loss, and no mortality; however, knockout of E glycosylation did not significantly affect neurovirulence. Mice immunized with the mutant virus developed a robust neutralizing antibody response and were completely protected from wild-type ZIKV challenge. In mosquitoes, the mutant virus exhibited diminished oral infectivity for the Aedes aegypti vector. Collectively, the results demonstrate that E glycosylation is critical for ZIKV infection of mammalian and mosquito hosts. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

DOE JGI Quality Metrics; Approaches to Scaling and Improving Metagenome Assembly (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

Energy Technology Data Exchange (ETDEWEB)

Copeland, Alex; Brown, C. Titus

2011-10-13

DOE JGI's Alex Copeland on "DOE JGI Quality Metrics" and Michigan State University's C. Titus Brown on "Approaches to Scaling and Improving Metagenome Assembly" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

High throughtput comparisons and profiling of metagenomes for industrially relevant enzymes

KAUST Repository

Alam, Intikhab

2016-01-26

More and more genomes and metagenomes are being sequenced since the advent of Next Generation Sequencing Technologies (NGS). Many metagenomic samples are collected from a variety of environments, each exhibiting a different environmental profile, e.g. temperature, environmental chemistry, etc… These metagenomes can be profiled to unearth enzymes relevant to several industries based on specific enzyme properties such as ability to work on extreme conditions, such as extreme temperatures, salinity, anaerobically, etc.. In this work, we present the DMAP platform comprising of a high-throughput metagenomic annotation pipeline and a data-warehouse for comparisons and profiling across large number of metagenomes. We developed two reference databases for profiling of important genes, one containing enzymes related to different industries and the other containing genes with potential bioactivity roles. In this presentation we describe an example analysis of a large number of publicly available metagenomic sample from TARA oceans study (Science 2015) that covers significant part of world oceans.

Marine Metagenome as A Resource for Novel Enzymes

KAUST Repository

Alma’abadi, Amani D.

2015-11-10

More than 99% of identified prokaryotes, including many from the marine environment, cannot be cultured in the laboratory. This lack of capability restricts our knowledge of microbial genetics and community ecology. Metagenomics, the culture-independent cloning of environmental DNAs that are isolated directly from an environmental sample, has already provided a wealth of information about the uncultured microbial world. It has also facilitated the discovery of novel biocatalysts by allowing researchers to probe directly into a huge diversity of enzymes within natural microbial communities. Recent advances in these studies have led to great interest in recruiting microbial enzymes for the development of environmentally-friendly industry. Although the metagenomics approach has many limitations, it is expected to provide not only scientific insights but also economic benefits, especially in industry. This review highlights the importance of metagenomics in mining microbial lipases, as an example, by using high-throughput techniques. In addition, we discuss challenges in the metagenomics as an important part of bioinformatics analysis in big data.

Importance of N-Glycosylation on CD147 for Its Biological Functions

Science.gov (United States)

Bai, Yang; Huang, Wan; Ma, Li-Tian; Jiang, Jian-Li; Chen, Zhi-Nan

2014-01-01

Glycosylation of glycoproteins is one of many molecular changes that accompany malignant transformation. Post-translational modifications of proteins are closely associated with the adhesion, invasion, and metastasis of tumor cells. CD147, a tumor-associated antigen that is highly expressed on the cell surface of various tumors, is a potential target for cancer diagnosis and therapy. A significant biochemical property of CD147 is its high level of glycosylation. Studies on the structure and function of CD147 glycosylation provide valuable clues to the development of targeted therapies for cancer. Here, we review current understanding of the glycosylation characteristics of CD147 and the glycosyltransferases involved in the biosynthesis of CD147 N-glycans. Finally, we discuss proteins regulating CD147 glycosylation and the biological functions of CD147 glycosylation. PMID:24739808

Importance of N-Glycosylation on CD147 for Its Biological Functions

Directory of Open Access Journals (Sweden)

Yang Bai

2014-04-01

Full Text Available Glycosylation of glycoproteins is one of many molecular changes that accompany malignant transformation. Post-translational modifications of proteins are closely associated with the adhesion, invasion, and metastasis of tumor cells. CD147, a tumor-associated antigen that is highly expressed on the cell surface of various tumors, is a potential target for cancer diagnosis and therapy. A significant biochemical property of CD147 is its high level of glycosylation. Studies on the structure and function of CD147 glycosylation provide valuable clues to the development of targeted therapies for cancer. Here, we review current understanding of the glycosylation characteristics of CD147 and the glycosyltransferases involved in the biosynthesis of CD147 N-glycans. Finally, we discuss proteins regulating CD147 glycosylation and the biological functions of CD147 glycosylation.

Bracken: estimating species abundance in metagenomics data

Directory of Open Access Journals (Sweden)

Jennifer Lu

2017-01-01

Full Text Available Metagenomic experiments attempt to characterize microbial communities using high-throughput DNA sequencing. Identification of the microorganisms in a sample provides information about the genetic profile, population structure, and role of microorganisms within an environment. Until recently, most metagenomics studies focused on high-level characterization at the level of phyla, or alternatively sequenced the 16S ribosomal RNA gene that is present in bacterial species. As the cost of sequencing has fallen, though, metagenomics experiments have increasingly used unbiased shotgun sequencing to capture all the organisms in a sample. This approach requires a method for estimating abundance directly from the raw read data. Here we describe a fast, accurate new method that computes the abundance at the species level using the reads collected in a metagenomics experiment. Bracken (Bayesian Reestimation of Abundance after Classification with KrakEN uses the taxonomic assignments made by Kraken, a very fast read-level classifier, along with information about the genomes themselves to estimate abundance at the species level, the genus level, or above. We demonstrate that Bracken can produce accurate species- and genus-level abundance estimates even when a sample contains multiple near-identical species.

Links between CD147 Function, Glycosylation, and Caveolin-1

OpenAIRE

Tang, Wei; Chang, Sharon B.; Hemler, Martin E.

2004-01-01

Cell surface CD147 shows remarkable variations in size (31-65 kDa) because of heterogeneous N-glycosylation, with the most highly glycosylated forms functioning to induce matrix metalloproteinase (MMP) production. Here we show that all three CD147 N-glycosylation sites make similar contributions to both high and low glycoforms (HG- and LG-CD147). l-Phytohemagglutinin lectin binding and swainsonine inhibition experiments indicated that HG-CD147 contains N-acetylglucosaminyltransferase V-cataly...

MetaQUAST: evaluation of metagenome assemblies.

Science.gov (United States)

Mikheenko, Alla; Saveliev, Vladislav; Gurevich, Alexey

2016-04-01

During the past years we have witnessed the rapid development of new metagenome assembly methods. Although there are many benchmark utilities designed for single-genome assemblies, there is no well-recognized evaluation and comparison tool for metagenomic-specific analogues. In this article, we present MetaQUAST, a modification of QUAST, the state-of-the-art tool for genome assembly evaluation based on alignment of contigs to a reference. MetaQUAST addresses such metagenome datasets features as (i) unknown species content by detecting and downloading reference sequences, (ii) huge diversity by giving comprehensive reports for multiple genomes and (iii) presence of highly relative species by detecting chimeric contigs. We demonstrate MetaQUAST performance by comparing several leading assemblers on one simulated and two real datasets. http://bioinf.spbau.ru/metaquast aleksey.gurevich@spbu.ru Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

N-linked glycosylation of the immunoglobulin variable region

NARCIS (Netherlands)

van de Bovenkamp, Fleur S.; Derksen, Ninotska I. L.; Ooijevaar-de Heer, Pleuni; van Schie, Karin A.; Kruithof, Simone; Berkowska, Magdalena A.; van der Schoot, C. Ellen; Ijspeert, Hanna; van der Burg, Mirjam; Gils, Ann; Hafkenscheid, Lise; Toes, René E. M.; Rombouts, Yoann; Plomp, Rosina; Wuhrer, Manfred; van Ham, S. Marieke; Vidarsson, Gestur; Rispens, Theo

2018-01-01

N-glycosylation sites are introduced at positions in which glycans can affect antigen binding as a result of a specific clustering of progenitor glycosylation sites in the germline sequences of variable domain genes. By analyzing multiple human monoclonal and polyclonal (auto)antibody responses, we

Metagenomics and Bioinformatics in Microbial Ecology: Current Status and Beyond.

Science.gov (United States)

Hiraoka, Satoshi; Yang, Ching-Chia; Iwasaki, Wataru

2016-09-29

Metagenomic approaches are now commonly used in microbial ecology to study microbial communities in more detail, including many strains that cannot be cultivated in the laboratory. Bioinformatic analyses make it possible to mine huge metagenomic datasets and discover general patterns that govern microbial ecosystems. However, the findings of typical metagenomic and bioinformatic analyses still do not completely describe the ecology and evolution of microbes in their environments. Most analyses still depend on straightforward sequence similarity searches against reference databases. We herein review the current state of metagenomics and bioinformatics in microbial ecology and discuss future directions for the field. New techniques will allow us to go beyond routine analyses and broaden our knowledge of microbial ecosystems. We need to enrich reference databases, promote platforms that enable meta- or comprehensive analyses of diverse metagenomic datasets, devise methods that utilize long-read sequence information, and develop more powerful bioinformatic methods to analyze data from diverse perspectives.

FY11 Report on Metagenome Analysis using Pathogen Marker Libraries

Energy Technology Data Exchange (ETDEWEB)

Gardner, Shea N. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Allen, Jonathan E. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); McLoughlin, Kevin S. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Slezak, Tom [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

2011-06-02

A method, sequence library, and software suite was invented to rapidly assess whether any member of a pre-specified list of threat organisms or their near neighbors is present in a metagenome. The system was designed to handle mega- to giga-bases of FASTA-formatted raw sequence reads from short or long read next generation sequencing platforms. The approach is to pre-calculate a viral and a bacterial "Pathogen Marker Library" (PML) containing sub-sequences specific to pathogens or their near neighbors. A list of expected matches comparing every bacterial or viral genome against the PML sequences is also pre-calculated. To analyze a metagenome, reads are compared to the PML, and observed PML-metagenome matches are compared to the expected PML-genome matches, and the ratio of observed relative to expected matches is reported. In other words, a 3-way comparison among the PML, metagenome, and existing genome sequences is used to quickly assess which (if any) species included in the PML is likely to be present in the metagenome, based on available sequence data. Our tests showed that the species with the most PML matches correctly indicated the organism sequenced for empirical metagenomes consisting of a cultured, relatively pure isolate. These runs completed in 1 minute to 3 hours on 12 CPU (1 thread/CPU), depending on the metagenome and PML. Using more threads on the same number of CPU resulted in speed improvements roughly proportional to the number of threads. Simulations indicated that detection sensitivity depends on both sequencing coverage levels for a species and the size of the PML: species were correctly detected even at ~0.003x coverage by the large PMLs, and at ~0.03x coverage by the smaller PMLs. Matches to true positive species were 3-4 orders of magnitude higher than to false positives. Simulations with short reads (36 nt and ~260 nt) showed that species were usually detected for metagenome coverage above 0.005x and coverage in the PML above 0.05x, and

MetaStorm: A Public Resource for Customizable Metagenomics Annotation.

Directory of Open Access Journals (Sweden)

Gustavo Arango-Argoty

Full Text Available Metagenomics is a trending research area, calling for the need to analyze large quantities of data generated from next generation DNA sequencing technologies. The need to store, retrieve, analyze, share, and visualize such data challenges current online computational systems. Interpretation and annotation of specific information is especially a challenge for metagenomic data sets derived from environmental samples, because current annotation systems only offer broad classification of microbial diversity and function. Moreover, existing resources are not configured to readily address common questions relevant to environmental systems. Here we developed a new online user-friendly metagenomic analysis server called MetaStorm (http://bench.cs.vt.edu/MetaStorm/, which facilitates customization of computational analysis for metagenomic data sets. Users can upload their own reference databases to tailor the metagenomics annotation to focus on various taxonomic and functional gene markers of interest. MetaStorm offers two major analysis pipelines: an assembly-based annotation pipeline and the standard read annotation pipeline used by existing web servers. These pipelines can be selected individually or together. Overall, MetaStorm provides enhanced interactive visualization to allow researchers to explore and manipulate taxonomy and functional annotation at various levels of resolution.

MetaStorm: A Public Resource for Customizable Metagenomics Annotation.

Science.gov (United States)

Arango-Argoty, Gustavo; Singh, Gargi; Heath, Lenwood S; Pruden, Amy; Xiao, Weidong; Zhang, Liqing

2016-01-01

Metagenomics is a trending research area, calling for the need to analyze large quantities of data generated from next generation DNA sequencing technologies. The need to store, retrieve, analyze, share, and visualize such data challenges current online computational systems. Interpretation and annotation of specific information is especially a challenge for metagenomic data sets derived from environmental samples, because current annotation systems only offer broad classification of microbial diversity and function. Moreover, existing resources are not configured to readily address common questions relevant to environmental systems. Here we developed a new online user-friendly metagenomic analysis server called MetaStorm (http://bench.cs.vt.edu/MetaStorm/), which facilitates customization of computational analysis for metagenomic data sets. Users can upload their own reference databases to tailor the metagenomics annotation to focus on various taxonomic and functional gene markers of interest. MetaStorm offers two major analysis pipelines: an assembly-based annotation pipeline and the standard read annotation pipeline used by existing web servers. These pipelines can be selected individually or together. Overall, MetaStorm provides enhanced interactive visualization to allow researchers to explore and manipulate taxonomy and functional annotation at various levels of resolution.

MetaStorm: A Public Resource for Customizable Metagenomics Annotation

Science.gov (United States)

Arango-Argoty, Gustavo; Singh, Gargi; Heath, Lenwood S.; Pruden, Amy; Xiao, Weidong; Zhang, Liqing

2016-01-01

Metagenomics is a trending research area, calling for the need to analyze large quantities of data generated from next generation DNA sequencing technologies. The need to store, retrieve, analyze, share, and visualize such data challenges current online computational systems. Interpretation and annotation of specific information is especially a challenge for metagenomic data sets derived from environmental samples, because current annotation systems only offer broad classification of microbial diversity and function. Moreover, existing resources are not configured to readily address common questions relevant to environmental systems. Here we developed a new online user-friendly metagenomic analysis server called MetaStorm (http://bench.cs.vt.edu/MetaStorm/), which facilitates customization of computational analysis for metagenomic data sets. Users can upload their own reference databases to tailor the metagenomics annotation to focus on various taxonomic and functional gene markers of interest. MetaStorm offers two major analysis pipelines: an assembly-based annotation pipeline and the standard read annotation pipeline used by existing web servers. These pipelines can be selected individually or together. Overall, MetaStorm provides enhanced interactive visualization to allow researchers to explore and manipulate taxonomy and functional annotation at various levels of resolution. PMID:27632579

[The role of protein glycosylation in immune system].

Science.gov (United States)

Ząbczyńska, Marta; Pocheć, Ewa

2015-01-01

Glycosylation is one of the most frequent post-translational modifications of proteins. The majority of cell surface and secreted proteins involved in immune response is glycosylated. The structural diversity of glycans depends on monosaccharide composition, type of glycosidic linkage and branching. These structural modifications determine a great variability of glycoproteins. The oligosaccharide components of proteins are regulated mostly by expression of glycosyltransferases and glycosidases and many environmental factors. Glycosylation influences the function of all immune cells. Glycans play a crucial role in intercellular contacts and leukocytes migration. These interactions are important in activation and proliferation of leukocytes and during immune response. The key immune proteins, such as TCR, MHC, TLR and antibodies are glycosylated. Sugars on the surface of pathogens and self-surface glycoproteins are recognized by special carbohydrate binding proteins called lectins. Changes of glycan structure are common in many pathological processes occurring in immune system, therefore they are used as molecular markers of different diseases.

Hydrophobic Man-1-P derivatives correct abnormal glycosylation in Type I congenital disorder of glycosylation fibroblasts.

Science.gov (United States)

Eklund, Erik A; Merbouh, Nabyl; Ichikawa, Mie; Nishikawa, Atsushi; Clima, Jessica M; Dorman, James A; Norberg, Thomas; Freeze, Hudson H

2005-11-01

Patients with Type I congenital disorders of glycosylation (CDG-I) make incomplete lipid-linked oligosaccharides (LLO). These glycans are poorly transferred to proteins resulting in unoccupied glycosylation sequons. Mutations in phosphomannomutase (PMM2) cause CDG-Ia by reducing the activity of PMM, which converts mannose (Man)-6-P to Man-1-P before formation of GDP-Man. These patients have reduced Man-1-P and GDP-Man. To replenish intracellular Man-1-P pools in CDG-Ia cells, we synthesized two hydrophobic, membrane permeable acylated versions of Man-1-P and determined their ability to normalize LLO size and N-glycosylation in CDG-Ia fibroblasts. Both compounds, compound I (diacetoxymethyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl phosphate) (C-I) and compound II (diacetoxymethyl 2,3,4,6-tetra-O-ethyloxycarbonyl-alpha-D-mannopyranosyl phosphate) (C-II), contain two acetoxymethyl (CH2OAc) groups O-linked to phosphorous. C-I contains acetyl esters and C-II contains ethylcarbonate (CO2Et) esters on the Man residue. Both C-I and C-II normalized truncated LLO, but C-II was about 2-fold more efficient than C-I. C-II replenished the GDP-Man pool in CDG-Ia cells and was more efficiently incorporated into glycoproteins than exogenous Man at low concentrations (25-75 mM). In a glycosylation assay of DNaseI in CDG-Ia cells, C-II restored glycosylation to control cell levels. C-II also corrected impaired LLO biosynthesis in cells from a Dolichol (Dol)-P-Man deficient patient (CDG-Ie) and partially corrected LLO in cells from an ALG12 mannosyltransferase-deficient patient (CDG-Ig), whereas cells from an ALG3-deficient patient (CDG-Id) and from an MPDU1-deficient patient (CDG-If) were not corrected. These results validate the general concept of using pro-Man-1-P substrates as potential therapeutics for CDG-I patients.

Prion propagation in cells expressing PrP glycosylation mutants.

Science.gov (United States)

Salamat, Muhammad K; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

2011-04-01

Infection by prions involves conversion of a host-encoded cell surface protein (PrP(C)) to a disease-related isoform (PrP(Sc)). PrP(C) carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrP(C) glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrP(Sc) and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrP(Sc), while PrP(C) with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrP(C), were able to form infectious PrP(Sc). Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection.

Unusual glycosylation of proteins: Beyond the universal sequon and other amino acids.

Science.gov (United States)

Dutta, Devawati; Mandal, Chhabinath; Mandal, Chitra

2017-12-01

Glycosylation of proteins is the most common, multifaceted co- and post-translational modification responsible for many biological processes and cellular functions. Significant alterations and aberrations of these processes are related to various pathological conditions, and often turn out to be disease biomarkers. Conventional N-glycosylation occurs through the recognition of the consensus sequon, asparagine (Asn)-X-serine (Ser)/threonine (Thr), where X is any amino acid except for proline, with N-acetylglucosamine (GlcNAc) as the first glycosidic linkage. Usually, O-glycosylation adds a glycan to the hydroxyl group of Ser or Thr beginning with N-acetylgalactosamine (GalNAc). Protein glycosylation is further governed by additional diversifications in sequon and structure, which are yet to be fully explored. This review mainly focuses on the occurrence of N-glycosylation in non-consensus motifs, where Ser/Thr at the +2 position is substituted by other amino acids. Additionally, N-glycosylation is also observed in other amide/amine group-containing amino acids. Similarly, O-glycosylation occurs at hydroxyl group-containing amino acids other than serine/threonine. The neighbouring amino acids and local structural features around the potential glycosylation site also play a significant role in determining the extent of glycosylation. All of these phenomena that yield glycosylation at the atypical sites are reported in a variety of biological systems, including different pathological conditions. Therefore, the discovery of more novel sequence patterns for N- and O-glycosylation may help in understanding the functions of complex biological processes and cellular functions. Taken together, all these information provided in this review would be helpful for the biological readers. Copyright © 2017 Elsevier B.V. All rights reserved.

Computational workflow for the fine-grained analysis of metagenomic samples.

Science.gov (United States)

Pérez-Wohlfeil, Esteban; Arjona-Medina, Jose A; Torreno, Oscar; Ulzurrun, Eugenia; Trelles, Oswaldo

2016-10-25

The field of metagenomics, defined as the direct genetic analysis of uncultured samples of genomes contained within an environmental sample, is gaining increasing popularity. The aim of studies of metagenomics is to determine the species present in an environmental community and identify changes in the abundance of species under different conditions. Current metagenomic analysis software faces bottlenecks due to the high computational load required to analyze complex samples. A computational open-source workflow has been developed for the detailed analysis of metagenomes. This workflow provides new tools and datafile specifications that facilitate the identification of differences in abundance of reads assigned to taxa (mapping), enables the detection of reads of low-abundance bacteria (producing evidence of their presence), provides new concepts for filtering spurious matches, etc. Innovative visualization ideas for improved display of metagenomic diversity are also proposed to better understand how reads are mapped to taxa. Illustrative examples are provided based on the study of two collections of metagenomes from faecal microbial communities of adult female monozygotic and dizygotic twin pairs concordant for leanness or obesity and their mothers. The proposed workflow provides an open environment that offers the opportunity to perform the mapping process using different reference databases. Additionally, this workflow shows the specifications of the mapping process and datafile formats to facilitate the development of new plugins for further post-processing. This open and extensible platform has been designed with the aim of enabling in-depth analysis of metagenomic samples and better understanding of the underlying biological processes.

Computational workflow for the fine-grained analysis of metagenomic samples

Directory of Open Access Journals (Sweden)

Esteban Pérez-Wohlfeil

2016-10-01

Full Text Available Abstract Background The field of metagenomics, defined as the direct genetic analysis of uncultured samples of genomes contained within an environmental sample, is gaining increasing popularity. The aim of studies of metagenomics is to determine the species present in an environmental community and identify changes in the abundance of species under different conditions. Current metagenomic analysis software faces bottlenecks due to the high computational load required to analyze complex samples. Results A computational open-source workflow has been developed for the detailed analysis of metagenomes. This workflow provides new tools and datafile specifications that facilitate the identification of differences in abundance of reads assigned to taxa (mapping, enables the detection of reads of low-abundance bacteria (producing evidence of their presence, provides new concepts for filtering spurious matches, etc. Innovative visualization ideas for improved display of metagenomic diversity are also proposed to better understand how reads are mapped to taxa. Illustrative examples are provided based on the study of two collections of metagenomes from faecal microbial communities of adult female monozygotic and dizygotic twin pairs concordant for leanness or obesity and their mothers. Conclusions The proposed workflow provides an open environment that offers the opportunity to perform the mapping process using different reference databases. Additionally, this workflow shows the specifications of the mapping process and datafile formats to facilitate the development of new plugins for further post-processing. This open and extensible platform has been designed with the aim of enabling in-depth analysis of metagenomic samples and better understanding of the underlying biological processes.

Marine Metagenome as A Resource for Novel Enzymes

Directory of Open Access Journals (Sweden)

Amani D. Alma’abadi

2015-10-01

Full Text Available More than 99% of identified prokaryotes, including many from the marine environment, cannot be cultured in the laboratory. This lack of capability restricts our knowledge of microbial genetics and community ecology. Metagenomics, the culture-independent cloning of environmental DNAs that are isolated directly from an environmental sample, has already provided a wealth of information about the uncultured microbial world. It has also facilitated the discovery of novel biocatalysts by allowing researchers to probe directly into a huge diversity of enzymes within natural microbial communities. Recent advances in these studies have led to a great interest in recruiting microbial enzymes for the development of environmentally-friendly industry. Although the metagenomics approach has many limitations, it is expected to provide not only scientific insights but also economic benefits, especially in industry. This review highlights the importance of metagenomics in mining microbial lipases, as an example, by using high-throughput techniques. In addition, we discuss challenges in the metagenomics as an important part of bioinformatics analysis in big data.

Functional Metagenomic Investigations of the Human Intestinal Microbiota

Directory of Open Access Journals (Sweden)

Aimee Marguerite Moore

2011-10-01

Full Text Available The human intestinal microbiota encode multiple critical functions impacting human health, including, metabolism of dietary substrate, prevention of pathogen invasion, immune system modulation, and provision of a reservoir of antibiotic resistance genes accessible to pathogens. The complexity of this microbial community, its recalcitrance to standard cultivation and the immense diversity of its encoded genes has necessitated the development of novel molecular, microbiological, and genomic tools. Functional metagenomics is one such culture-independent technique used for decades to study environmental microorganisms but relatively recently applied to the study of the human commensal microbiota. Metagenomic functional screens characterize the functional capacity of a microbial community independent of identity to known genes by subjecting the metagenome to functional assays in a genetically tractable host. Here we highlight recent work applying this technique to study the functional diversity of the intestinal microbiota, and discuss how an approach combining high-throughput sequencing, cultivation, and metagenomic functional screens can improve our understanding of interactions between this complex community and its human host.

Functional Metagenomic Investigations of the Human Intestinal Microbiota

DEFF Research Database (Denmark)

Moore, Aimee M.; Munck, Christian; Sommer, Morten Otto Alexander

2011-01-01

The human intestinal microbiota encode multiple critical functions impacting human health, including metabolism of dietary substrate, prevention of pathogen invasion, immune system modulation, and provision of a reservoir of antibiotic resistance genes accessible to pathogens. The complexity...... microorganisms, but relatively recently applied to the study of the human commensal microbiota. Metagenomic functional screens characterize the functional capacity of a microbial community, independent of identity to known genes, by subjecting the metagenome to functional assays in a genetically tractable host....... Here we highlight recent work applying this technique to study the functional diversity of the intestinal microbiota, and discuss how an approach combining high-throughput sequencing, cultivation, and metagenomic functional screens can improve our understanding of interactions between this complex...

The role of glycosylation in breast cancer metastasis and cancer control

Directory of Open Access Journals (Sweden)

Alexandra eKölbl

2015-10-01

Full Text Available AbstractGlycosylation and its correlation to the formation of remote metastasis in breast cancer had been an important scientific topic in the last 25 years. With the development of new analytical techniques new insights were gained on the mechanisms underlying metastasis formation and the role of aberrant glycosylation within. Mucin-1 and Galectin were recognized as key players in glycosylation. Interestingly, aberrant carbohydrate structures seem to support the development of brain metastasis in breast cancer patients, as changes in glycosylation structures facilitate an overcoming of blood-brain barrier. Changes in the gene expression of glycosyltransferases are the leading cause for a modification of carbohydrate chains, so that also altered gene expression plays a role for glycosylation. In consequence, glycosylation and changes within can be useful for cancer diagnosis, determination of tumour stage and prognosis, but can as well be targets for therapeutic strategies. Thus, further research on this topic would worth wile for cancer combating.

WebMGA: a customizable web server for fast metagenomic sequence analysis.

Science.gov (United States)

Wu, Sitao; Zhu, Zhengwei; Fu, Liming; Niu, Beifang; Li, Weizhong

2011-09-07

The new field of metagenomics studies microorganism communities by culture-independent sequencing. With the advances in next-generation sequencing techniques, researchers are facing tremendous challenges in metagenomic data analysis due to huge quantity and high complexity of sequence data. Analyzing large datasets is extremely time-consuming; also metagenomic annotation involves a wide range of computational tools, which are difficult to be installed and maintained by common users. The tools provided by the few available web servers are also limited and have various constraints such as login requirement, long waiting time, inability to configure pipelines etc. We developed WebMGA, a customizable web server for fast metagenomic analysis. WebMGA includes over 20 commonly used tools such as ORF calling, sequence clustering, quality control of raw reads, removal of sequencing artifacts and contaminations, taxonomic analysis, functional annotation etc. WebMGA provides users with rapid metagenomic data analysis using fast and effective tools, which have been implemented to run in parallel on our local computer cluster. Users can access WebMGA through web browsers or programming scripts to perform individual analysis or to configure and run customized pipelines. WebMGA is freely available at http://weizhongli-lab.org/metagenomic-analysis. WebMGA offers to researchers many fast and unique tools and great flexibility for complex metagenomic data analysis.

WebMGA: a customizable web server for fast metagenomic sequence analysis

Directory of Open Access Journals (Sweden)

Niu Beifang

2011-09-01

Full Text Available Abstract Background The new field of metagenomics studies microorganism communities by culture-independent sequencing. With the advances in next-generation sequencing techniques, researchers are facing tremendous challenges in metagenomic data analysis due to huge quantity and high complexity of sequence data. Analyzing large datasets is extremely time-consuming; also metagenomic annotation involves a wide range of computational tools, which are difficult to be installed and maintained by common users. The tools provided by the few available web servers are also limited and have various constraints such as login requirement, long waiting time, inability to configure pipelines etc. Results We developed WebMGA, a customizable web server for fast metagenomic analysis. WebMGA includes over 20 commonly used tools such as ORF calling, sequence clustering, quality control of raw reads, removal of sequencing artifacts and contaminations, taxonomic analysis, functional annotation etc. WebMGA provides users with rapid metagenomic data analysis using fast and effective tools, which have been implemented to run in parallel on our local computer cluster. Users can access WebMGA through web browsers or programming scripts to perform individual analysis or to configure and run customized pipelines. WebMGA is freely available at http://weizhongli-lab.org/metagenomic-analysis. Conclusions WebMGA offers to researchers many fast and unique tools and great flexibility for complex metagenomic data analysis.

Metagenomics at Grass Roots

Indian Academy of Sciences (India)

Metagenomics is a robust, interdisciplinary approach for studyingmicrobial community composition, function, and dynamics.It typically involves a core of molecular biology, microbiology,ecology, statistics, and computational biology. Excitingoutcomes anticipated from these studies include unravelingof complex interactions ...

Glycosylation Helps Cellulase Enzymes Bind to Plant Cell Walls (Fact Sheet)

Energy Technology Data Exchange (ETDEWEB)

2012-06-01

Computer simulations suggest a new strategy to design enhanced enzymes for biofuels production. Large-scale computer simulations predict that the addition of glycosylation on carbohydrate-binding modules can dramatically improve the binding affinity of these protein domains over amino acid mutations alone. These simulations suggest that glycosylation can be used as a protein engineering tool to enhance the activity of cellulase enzymes, which are a key component in the conversion of cellulose to soluble sugars in the production of biofuels. Glycosylation is the covalent attachment of carbohydrate molecules to protein side chains, and is present in many proteins across all kingdoms of life. Moreover, glycosylation is known to serve a wide variety of functions in biological recognition, cell signaling, and metabolism. Cellulase enzymes, which are responsible for deconstructing cellulose found in plant cell walls to glucose, contain glycosylation that when modified can affect enzymatic activity-often in an unpredictable manner. To gain insight into the role of glycosylation on cellulase activity, scientists at the National Renewable Energy Laboratory (NREL) used computer simulation to predict that adding glycosylation on the carbohydrate-binding module of a cellulase enzyme dramatically boosts the binding affinity to cellulose-more than standard protein engineering approaches in which amino acids are mutated. Because it is known that higher binding affinity in cellulases leads to higher activity, this work suggests a new route to designing enhanced enzymes for biofuels production. More generally, this work suggests that tuning glycosylation in cellulase enzymes is a key factor to consider when engineering biochemical conversion processes, and that more work is needed to understand how glycosylation affects cellulase activity at the molecular level.

Metagenomics at Grass Roots

Indian Academy of Sciences (India)

CAMERA (Community Cyber-infrastructure for Advanced Mi- crobial Ecology .... Acidobacteria known to metabolize a variety of car- bon sources .... [7] J Nesme et al., Back to the future of soil metagenomics, Frontiers in Microbi- ology, Vol.7 ...

Unlocking the potential of metagenomics through replicated experimental design

NARCIS (Netherlands)

Knight, R.; Jansson, J.; Field, D.; Fierer, N.; Desai, N.; Fuhrman, J.A.; Hugenholtz, P.; Van der Lelie, D.; Meyer, F.; Stevens, R.; Bailey, M.J.; Gordon, J.I.; Kowalchuk, G.A.; Gilbert, J.A.

2012-01-01

Metagenomics holds enormous promise for discovering novel enzymes and organisms that are biomarkers or drivers of processes relevant to disease, industry and the environment. In the past two years, we have seen a paradigm shift in metagenomics to the application of cross-sectional and longitudinal

Unlocking the potential of metagenomics through replicated experimental design.

NARCIS (Netherlands)

Knight, R.; Jansson, J.; Field, D.; Fierer, N.; Desai, N.; Fuhrman, J.A.; Hugenholtz, P.; van der Lelie, D.; Meyer, F.; Stevens, R.; Bailey, M.J.; Gordon, J.I.; Kowalchuk, G.A.; Gilbert, J.A.

2012-01-01

Metagenomics holds enormous promise for discovering novel enzymes and organisms that are biomarkers or drivers of processes relevant to disease, industry and the environment. In the past two years, we have seen a paradigm shift in metagenomics to the application of cross-sectional and longitudinal

Surface glycosylation profiles of urine extracellular vesicles.

Directory of Open Access Journals (Sweden)

Jared Q Gerlach

Full Text Available Urinary extracellular vesicles (uEVs are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications.

Metagenomics: The Next Culture-Independent Game Changer

Directory of Open Access Journals (Sweden)

Jessica D. Forbes

2017-07-01

Full Text Available A trend towards the abandonment of obtaining pure culture isolates in frontline laboratories is at a crossroads with the ability of public health agencies to perform their basic mandate of foodborne disease surveillance and response. The implementation of culture-independent diagnostic tests (CIDTs including nucleic acid and antigen-based assays for acute gastroenteritis is leaving public health agencies without laboratory evidence to link clinical cases to each other and to food or environmental substances. This limits the efficacy of public health epidemiology and surveillance as well as outbreak detection and investigation. Foodborne outbreaks have the potential to remain undetected or have insufficient evidence to support source attribution and may inadvertently increase the incidence of foodborne diseases. Next-generation sequencing of pure culture isolates in clinical microbiology laboratories has the potential to revolutionize the fields of food safety and public health. Metagenomics and other ‘omics’ disciplines could provide the solution to a cultureless future in clinical microbiology, food safety and public health. Data mining of information obtained from metagenomics assays can be particularly useful for the identification of clinical causative agents or foodborne contamination, detection of AMR and/or virulence factors, in addition to providing high-resolution subtyping data. Thus, metagenomics assays may provide a universal test for clinical diagnostics, foodborne pathogen detection, subtyping and investigation. This information has the potential to reform the field of enteric disease diagnostics and surveillance and also infectious diseases as a whole. The aim of this review will be to present the current state of CIDTs in diagnostic and public health laboratories as they relate to foodborne illness and food safety. Moreover, we will also discuss the diagnostic and subtyping utility and concomitant bias limitations of

Cross-cutting activities: Soil quality and soil metagenomics

OpenAIRE

Motavalli, Peter P.; Garrett, Karen A.

2008-01-01

This presentation reports on the work of the SANREM CRSP cross-cutting activities "Assessing and Managing Soil Quality for Sustainable Agricultural Systems" and "Soil Metagenomics to Construct Indicators of Soil Degradation." The introduction gives an overview of the extensiveness of soil degradation globally and defines soil quality. The objectives of the soil quality cross cutting activity are: CCRA-4 (Soil Metagenomics)

SPHINX--an algorithm for taxonomic binning of metagenomic sequences.

Science.gov (United States)

Mohammed, Monzoorul Haque; Ghosh, Tarini Shankar; Singh, Nitin Kumar; Mande, Sharmila S

2011-01-01

Compared with composition-based binning algorithms, the binning accuracy and specificity of alignment-based binning algorithms is significantly higher. However, being alignment-based, the latter class of algorithms require enormous amount of time and computing resources for binning huge metagenomic datasets. The motivation was to develop a binning approach that can analyze metagenomic datasets as rapidly as composition-based approaches, but nevertheless has the accuracy and specificity of alignment-based algorithms. This article describes a hybrid binning approach (SPHINX) that achieves high binning efficiency by utilizing the principles of both 'composition'- and 'alignment'-based binning algorithms. Validation results with simulated sequence datasets indicate that SPHINX is able to analyze metagenomic sequences as rapidly as composition-based algorithms. Furthermore, the binning efficiency (in terms of accuracy and specificity of assignments) of SPHINX is observed to be comparable with results obtained using alignment-based algorithms. A web server for the SPHINX algorithm is available at http://metagenomics.atc.tcs.com/SPHINX/.

Gene prediction in metagenomic fragments: A large scale machine learning approach

Directory of Open Access Journals (Sweden)

Morgenstern Burkhard

2008-04-01

Full Text Available Abstract Background Metagenomics is an approach to the characterization of microbial genomes via the direct isolation of genomic sequences from the environment without prior cultivation. The amount of metagenomic sequence data is growing fast while computational methods for metagenome analysis are still in their infancy. In contrast to genomic sequences of single species, which can usually be assembled and analyzed by many available methods, a large proportion of metagenome data remains as unassembled anonymous sequencing reads. One of the aims of all metagenomic sequencing projects is the identification of novel genes. Short length, for example, Sanger sequencing yields on average 700 bp fragments, and unknown phylogenetic origin of most fragments require approaches to gene prediction that are different from the currently available methods for genomes of single species. In particular, the large size of metagenomic samples requires fast and accurate methods with small numbers of false positive predictions. Results We introduce a novel gene prediction algorithm for metagenomic fragments based on a two-stage machine learning approach. In the first stage, we use linear discriminants for monocodon usage, dicodon usage and translation initiation sites to extract features from DNA sequences. In the second stage, an artificial neural network combines these features with open reading frame length and fragment GC-content to compute the probability that this open reading frame encodes a protein. This probability is used for the classification and scoring of gene candidates. With large scale training, our method provides fast single fragment predictions with good sensitivity and specificity on artificially fragmented genomic DNA. Additionally, this method is able to predict translation initiation sites accurately and distinguishes complete from incomplete genes with high reliability. Conclusion Large scale machine learning methods are well-suited for gene

A catalog of the mouse gut metagenome

DEFF Research Database (Denmark)

Xiao, Liang; Feng, Qiang; Liang, Suisha

2015-01-01

laboratories and fed either a low-fat or high-fat diet. Similar to the human gut microbiome, >99% of the cataloged genes are bacterial. We identified 541 metagenomic species and defined a core set of 26 metagenomic species found in 95% of the mice. The mouse gut microbiome is functionally similar to its human......We established a catalog of the mouse gut metagenome comprising ∼2.6 million nonredundant genes by sequencing DNA from fecal samples of 184 mice. To secure high microbiome diversity, we used mouse strains of diverse genetic backgrounds, from different providers, kept in different housing...... counterpart, with 95.2% of its Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologous groups in common. However, only 4.0% of the mouse gut microbial genes were shared (95% identity, 90% coverage) with those of the human gut microbiome. This catalog provides a useful reference for future studies....

Glucose-tolerant β-glucosidase retrieved from a Kusaya gravy metagenome.

Science.gov (United States)

Uchiyama, Taku; Yaoi, Katusro; Miyazaki, Kentaro

2015-01-01

β-glucosidases (BGLs) hydrolyze cello-oligosaccharides to glucose and play a crucial role in the enzymatic saccharification of cellulosic biomass. Despite their significance for the production of glucose, most identified BGLs are commonly inhibited by low (∼mM) concentrations of glucose. Therefore, BGLs that are insensitive to glucose inhibition have great biotechnological merit. We applied a metagenomic approach to screen for such rare glucose-tolerant BGLs. A metagenomic library was created in Escherichia coli (∼10,000 colonies) and grown on LB agar plates containing 5-bromo-4-chloro-3-indolyl-β-D-glucoside, yielding 828 positive (blue) colonies. These were then arrayed in 96-well plates, grown in LB, and secondarily screened for activity in the presence of 10% (w/v) glucose. Seven glucose-tolerant clones were identified, each of which contained a single bgl gene. The genes were classified into two groups, differing by two nucleotides. The deduced amino acid sequences of these genes were identical (452 aa) and found to belong to the glycosyl hydrolase family 1. The recombinant protein (Ks5A7) was overproduced in E. coli as a C-terminal 6 × His-tagged protein and purified to apparent homogeneity. The molecular mass of the purified Ks5A7 was determined to be 54 kDa by SDS-PAGE, and 160 kDa by gel filtration analysis. The enzyme was optimally active at 45°C and pH 5.0-6.5 and retained full or 1.5-2-fold enhanced activity in the presence of 0.1-0.5 M glucose. It had a low KM (78 μM with p-nitrophenyl β-D-glucoside; 0.36 mM with cellobiose) and high V max (91 μmol min(-1) mg(-1) with p-nitrophenyl β-D-glucoside; 155 μmol min(-1) mg(-1) with cellobiose) among known glucose-tolerant BGLs and was free from substrate (0.1 M cellobiose) inhibition. The efficient use of Ks5A7 in conjunction with Trichoderma reesei cellulases in enzymatic saccharification of alkaline-treated rice straw was demonstrated by increased production of glucose.

Quantifying risk of penile prosthesis infection with elevated glycosylated hemoglobin.

Science.gov (United States)

Wilson, S K; Carson, C C; Cleves, M A; Delk, J R

1998-05-01

Elevation of glycosylated hemoglobin above levels of 11.5 mg.% has been considered a contraindication to penile prosthesis implantation in diabetic patients. We determine the predictive value of glycosylated hemoglobin A1C in penile prosthesis infections in diabetic and nondiabetic patients to confirm or deny this prevalent opinion. We conducted a 2-year prospective study of 389 patients, including 114 diabetics, who underwent 3-piece penile prosthesis implantation. All patients had similar preoperative preparation without regard to diabetic status, control or glycosylated hemoglobin A1C level. Risk of infection was statistically analyzed for diabetics versus nondiabetics, glycosylated hemoglobin A1C values above and below 11.5 mg.%, insulin dependent versus oral medication diabetics, and fasting blood sugars above and below 180 mg.%. Prosthesis infections developed in 10 diabetics (8.7%) and 11 nondiabetics (4.0%). No increased infection rate was observed in diabetics with high fasting sugars or diabetics on insulin. There was no statistically significant increased infection risk with increased levels of glycosylated hemoglobin A1C among all patients or among only the diabetics. In fact, there was no meaningful difference in the median or mean level of glycosylated hemoglobin A1C in the infected and noninfected patients regardless of diabetes. Use of glycosylated hemoglobin A1C values to identify and exclude surgical candidates with increased risk of infections is not proved by this study. Elevation of fasting sugar or insulin dependence also does not increase risk of infection in diabetics undergoing prosthesis implantation.

Method Development in the Regioselective Glycosylation of Unprotected Carbohydrates

DEFF Research Database (Denmark)

Niedbal, Dominika Alina

and the glycosylations were promoted by tetrabutylammonium bromide. The couplings were completely selective and gave rise to a number of 1,6-linked disaccharides with 1,2- cis-linked orientation. Project 2: Boron-mediated glycosylation of unprotected carbohydrates Boron-mediated regioselective Koenigs...

Functional metagenomics to decipher food-microbe-host crosstalk.

Science.gov (United States)

Larraufie, Pierre; de Wouters, Tomas; Potocki-Veronese, Gabrielle; Blottière, Hervé M; Doré, Joël

2015-02-01

The recent developments of metagenomics permit an extremely high-resolution molecular scan of the intestinal microbiota giving new insights and opening perspectives for clinical applications. Beyond the unprecedented vision of the intestinal microbiota given by large-scale quantitative metagenomics studies, such as the EU MetaHIT project, functional metagenomics tools allow the exploration of fine interactions between food constituents, microbiota and host, leading to the identification of signals and intimate mechanisms of crosstalk, especially between bacteria and human cells. Cloning of large genome fragments, either from complex intestinal communities or from selected bacteria, allows the screening of these biological resources for bioactivity towards complex plant polymers or functional food such as prebiotics. This permitted identification of novel carbohydrate-active enzyme families involved in dietary fibre and host glycan breakdown, and highlighted unsuspected bacterial players at the top of the intestinal microbial food chain. Similarly, exposure of fractions from genomic and metagenomic clones onto human cells engineered with reporter systems to track modulation of immune response, cell proliferation or cell metabolism has allowed the identification of bioactive clones modulating key cell signalling pathways or the induction of specific genes. This opens the possibility to decipher mechanisms by which commensal bacteria or candidate probiotics can modulate the activity of cells in the intestinal epithelium or even in distal organs such as the liver, adipose tissue or the brain. Hence, in spite of our inability to culture many of the dominant microbes of the human intestine, functional metagenomics open a new window for the exploration of food-microbe-host crosstalk.

New Hydrocarbon Degradation Pathways in the Microbial Metagenome from Brazilian Petroleum Reservoirs

Science.gov (United States)

Sierra-García, Isabel Natalia; Correa Alvarez, Javier; Pantaroto de Vasconcellos, Suzan; Pereira de Souza, Anete; dos Santos Neto, Eugenio Vaz; de Oliveira, Valéria Maia

2014-01-01

Current knowledge of the microbial diversity and metabolic pathways involved in hydrocarbon degradation in petroleum reservoirs is still limited, mostly due to the difficulty in recovering the complex community from such an extreme environment. Metagenomics is a valuable tool to investigate the genetic and functional diversity of previously uncultured microorganisms in natural environments. Using a function-driven metagenomic approach, we investigated the metabolic abilities of microbial communities in oil reservoirs. Here, we describe novel functional metabolic pathways involved in the biodegradation of aromatic compounds in a metagenomic library obtained from an oil reservoir. Although many of the deduced proteins shared homology with known enzymes of different well-described aerobic and anaerobic catabolic pathways, the metagenomic fragments did not contain the complete clusters known to be involved in hydrocarbon degradation. Instead, the metagenomic fragments comprised genes belonging to different pathways, showing novel gene arrangements. These results reinforce the potential of the metagenomic approach for the identification and elucidation of new genes and pathways in poorly studied environments and contribute to a broader perspective on the hydrocarbon degradation processes in petroleum reservoirs. PMID:24587220

Distribution of N-glycosylation sequons in proteins: how apart are they?

DEFF Research Database (Denmark)

Rao, Shyama Prasad; Buus, Ole Thomsen; Wollenweber, Bernd

2011-01-01

of experimentally confirmed eukaryotic N-glycoproteins we analyzed the relative position and distribution of sequons. N-Glycosylation probability was found to be lower in the termini of protein sequences compared to the mid region. N-glycosylated sequons were found much farther from C terminus compared to the N......N-glycosylation is a common protein modification process, which affects a number of properties of proteins. Little is known about the distribution of N-glycosylation sequons, for example, the distance between glycosylated sites and their position in the protein primary sequence. Using a large set......-terminus of the protein sequence and this effect was more pronounced for NXS sequons. The distribution of sequons, modeled based on balls-in-boxes classical occupancy, showed a near-maximum probability. Considerable proportion of sequons was found within a distance of ten amino acids, indicating that the steric hindrance...

Meta-IDBA: a de Novo assembler for metagenomic data.

Science.gov (United States)

Peng, Yu; Leung, Henry C M; Yiu, S M; Chin, Francis Y L

2011-07-01

Next-generation sequencing techniques allow us to generate reads from a microbial environment in order to analyze the microbial community. However, assembling of a set of mixed reads from different species to form contigs is a bottleneck of metagenomic research. Although there are many assemblers for assembling reads from a single genome, there are no assemblers for assembling reads in metagenomic data without reference genome sequences. Moreover, the performances of these assemblers on metagenomic data are far from satisfactory, because of the existence of common regions in the genomes of subspecies and species, which make the assembly problem much more complicated. We introduce the Meta-IDBA algorithm for assembling reads in metagenomic data, which contain multiple genomes from different species. There are two core steps in Meta-IDBA. It first tries to partition the de Bruijn graph into isolated components of different species based on an important observation. Then, for each component, it captures the slight variants of the genomes of subspecies from the same species by multiple alignments and represents the genome of one species, using a consensus sequence. Comparison of the performances of Meta-IDBA and existing assemblers, such as Velvet and Abyss for different metagenomic datasets shows that Meta-IDBA can reconstruct longer contigs with similar accuracy. Meta-IDBA toolkit is available at our website http://www.cs.hku.hk/~alse/metaidba. chin@cs.hku.hk.

Marine tunicates from Sangkarang Archipelago Indonesia: recent finding and bio-prospecting

Science.gov (United States)

Litaay, Magdalena

2018-03-01

Tunicate belongs to urochordata that inhabit benthic area of coral reefs. This paper attempts to compile result of several studies on diversity of marine tunicates from Sangkarang Archipelago of South Sulawesi Indonesia. Method for tunicates sample collection was line transect method that applied at two 3 and 7 m depth. A 50 m line transect was applied parallel to a shore line in each depth and was done in duplicate. A 2.5 m plot was places side by side of transect, in which all tunicate inside plot was counted, identified, and photographed, respectively. Tunicates identification was based on morphological characteristics. The latest finding shows that eighteen species of tunicates were recorded in Samalona waters as also the same number of species in Barrang Caddi waters. The result from this finding was compared to previous studies done at other part of Sangkarang area. This study concluded that this archipelago is rich in tunicates and these resources can be utilized for various purposes. Bioprospect of marine tunicates is also discus in the present study.

Prion Propagation in Cells Expressing PrP Glycosylation Mutants ▿

Science.gov (United States)

Salamat, Muhammad K.; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

2011-01-01

Infection by prions involves conversion of a host-encoded cell surface protein (PrPC) to a disease-related isoform (PrPSc). PrPC carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrPC glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrPSc and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrPSc, while PrPC with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrPC, were able to form infectious PrPSc. Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection. PMID:21248032

Effect of glycosylation on biodistribution of radiolabeled glucagon-like peptide 1

International Nuclear Information System (INIS)

Watanabe, Ayahisa; Nishijima, Ken-ichi; Zhao, Songji; Tamaki, Nagara; Kuge, Yuji; Tanaka, Yoshikazu; Itoh, Takeshi; Takemoto, Hiroshi

2012-01-01

Glycosylation is generally applicable as a strategy for increasing the activity of bioactive proteins. In this study, we examined the effect of glycosylation on biodistribution of radiolabeled glucagon-like peptide 1 (GLP-1) as a bioactive peptide for type 2 diabetes. Noninvasive imaging studies were performed using a gamma camera after the intravenous administration of 123 I-GLP-1 or 123 I-α2, 6-sialyl N-acetyllactosamine (glycosylated) GLP-1 in rats. In ex vivo biodistribution studies using 125 I-GLP-1 or 125 I-glycosylated GLP-1, organ samples were measured for radioactivity. Plasma samples were added to 15% trichloroacetic acid (TCA) to obtain TCA-insoluble and TCA-soluble fractions. The radioactivity in the TCA-insoluble and TCA-soluble fractions was measured. In the noninvasive imaging studies, a relatively high accumulation level of 123 I-GLP-1 was found in the liver, which is the major organ to eliminate exogenous GLP-1. The area under the time-activity curve (AUC) of 123 I-glycosylated GLP-1 in the liver was significantly lower (89%) than that of 123 I-GLP-1. These results were consistent with those of ex vivo biodistribution studies using 125 I-labeled peptides. The AUC of 125 I-glycosylated GLP-1 in the TCA-insoluble fraction was significantly higher (1.7-fold) than that of GLP-1. This study demonstrated that glycosylation significantly decreased the distribution of radiolabeled GLP-1 into the liver and increased the concentration of radiolabeled GLP-1 in plasma. These results suggested that glycosylation is a useful strategy for decreasing the distribution into the liver of bioactive peptides as desirable pharmaceuticals. (author)

Metagenomes from two microbial consortia associated with Santa Barbara seep oil.

Science.gov (United States)

Hawley, Erik R; Malfatti, Stephanie A; Pagani, Ioanna; Huntemann, Marcel; Chen, Amy; Foster, Brian; Copeland, Alexander; del Rio, Tijana Glavina; Pati, Amrita; Jansson, Janet R; Gilbert, Jack A; Tringe, Susannah Green; Lorenson, Thomas D; Hess, Matthias

2014-12-01

The metagenomes from two microbial consortia associated with natural oils seeping into the Pacific Ocean offshore the coast of Santa Barbara (California, USA) were determined to complement already existing metagenomes generated from microbial communities associated with hydrocarbons that pollute the marine ecosystem. This genomics resource article is the first of two publications reporting a total of four new metagenomes from oils that seep into the Santa Barbara Channel. Copyright © 2014 Elsevier B.V. All rights reserved.

Genetic variability of psychrotolerant Acidithiobacillus ferrivorans revealed by (meta)genomic analysis.

Science.gov (United States)

González, Carolina; Yanquepe, María; Cardenas, Juan Pablo; Valdes, Jorge; Quatrini, Raquel; Holmes, David S; Dopson, Mark

2014-11-01

Acidophilic microorganisms inhabit low pH environments such as acid mine drainage that is generated when sulfide minerals are exposed to air. The genome sequence of the psychrotolerant Acidithiobacillus ferrivorans SS3 was compared to a metagenome from a low temperature acidic stream dominated by an A. ferrivorans-like strain. Stretches of genomic DNA characterized by few matches to the metagenome, termed 'metagenomic islands', encoded genes associated with metal efflux and pH homeostasis. The metagenomic islands were enriched in mobile elements such as phage proteins, transposases, integrases and in one case, predicted to be flanked by truncated tRNAs. Cus gene clusters predicted to be involved in copper efflux and further Cus-like RND systems were predicted to be located in metagenomic islands and therefore, constitute part of the flexible gene complement of the species. Phylogenetic analysis of Cus clusters showed both lineage specificity within the Acidithiobacillus genus as well as niche specificity associated with an acidic environment. The metagenomic islands also contained a predicted copper efflux P-type ATPase system and a polyphosphate kinase potentially involved in polyphosphate mediated copper resistance. This study identifies genetic variability of low temperature acidophiles that likely reflects metal resistance selective pressures in the copper rich environment. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Metagenomics and the protein universe

Science.gov (United States)

Godzik, Adam

2011-01-01

Metagenomics sequencing projects have dramatically increased our knowledge of the protein universe and provided over one-half of currently known protein sequences; they have also introduced a much broader phylogenetic diversity into the protein databases. The full analysis of metagenomic datasets is only beginning, but it has already led to the discovery of thousands of new protein families, likely representing novel functions specific to given environments. At the same time, a deeper analysis of such novel families, including experimental structure determination of some representatives, suggests that most of them represent distant homologs of already characterized protein families, and thus most of the protein diversity present in the new environments are due to functional divergence of the known protein families rather than the emergence of new ones. PMID:21497084

Metagenomic Detection Methods in Biopreparedness Outbreak Scenarios

DEFF Research Database (Denmark)

Karlsson, Oskar Erik; Hansen, Trine; Knutsson, Rickard

2013-01-01

In the field of diagnostic microbiology, rapid molecular methods are critically important for detecting pathogens. With rapid and accurate detection, preventive measures can be put in place early, thereby preventing loss of life and further spread of a disease. From a preparedness perspective...... of a clinical sample, creating a metagenome, in a single week of laboratory work. As new technologies emerge, their dissemination and capacity building must be facilitated, and criteria for use, as well as guidelines on how to report results, must be established. This article focuses on the use of metagenomics...

Diagnostic accuracy of urinary prostate protein glycosylation profiling in prostatitis diagnosis.

Science.gov (United States)

Vermassen, Tijl; Van Praet, Charles; Poelaert, Filip; Lumen, Nicolaas; Decaestecker, Karel; Hoebeke, Piet; Van Belle, Simon; Rottey, Sylvie; Delanghe, Joris

2015-01-01

Although prostatitis is a common male urinary tract infection, clinical diagnosis of prostatitis is difficult. The developmental mechanism of prostatitis is not yet unraveled which led to the elaboration of various biomarkers. As changes in asparagine-linked-(N-)-glycosylation were observed between healthy volunteers (HV), patients with benign prostate hyperplasia and prostate cancer patients, a difference could exist in biochemical parameters and urinary N-glycosylation between HV and prostatitis patients. We therefore investigated if prostatic protein glycosylation could improve the diagnosis of prostatitis. Differences in serum and urine biochemical markers and in total urine N-glycosylation profile of prostatic proteins were determined between HV (N=66) and prostatitis patients (N=36). Additionally, diagnostic accuracy of significant biochemical markers and changes in N-glycosylation was assessed. Urinary white blood cell (WBC) count enabled discrimination of HV from prostatitis patients (Pprostatitis patients from HV (Pprostatitis patients compared to HV (Pprostatitis. Further research is required to unravel the developmental course of prostatic inflammation.

Comparative fecal metagenomics unveils unique functional capacity of the swine gut

Directory of Open Access Journals (Sweden)

Martinson John

2011-05-01

Full Text Available Abstract Background Uncovering the taxonomic composition and functional capacity within the swine gut microbial consortia is of great importance to animal physiology and health as well as to food and water safety due to the presence of human pathogens in pig feces. Nonetheless, limited information on the functional diversity of the swine gut microbiome is available. Results Analysis of 637, 722 pyrosequencing reads (130 megabases generated from Yorkshire pig fecal DNA extracts was performed to help better understand the microbial diversity and largely unknown functional capacity of the swine gut microbiome. Swine fecal metagenomic sequences were annotated using both MG-RAST and JGI IMG/M-ER pipelines. Taxonomic analysis of metagenomic reads indicated that swine fecal microbiomes were dominated by Firmicutes and Bacteroidetes phyla. At a finer phylogenetic resolution, Prevotella spp. dominated the swine fecal metagenome, while some genes associated with Treponema and Anareovibrio species were found to be exclusively within the pig fecal metagenomic sequences analyzed. Functional analysis revealed that carbohydrate metabolism was the most abundant SEED subsystem, representing 13% of the swine metagenome. Genes associated with stress, virulence, cell wall and cell capsule were also abundant. Virulence factors associated with antibiotic resistance genes with highest sequence homology to genes in Bacteroidetes, Clostridia, and Methanosarcina were numerous within the gene families unique to the swine fecal metagenomes. Other abundant proteins unique to the distal swine gut shared high sequence homology to putative carbohydrate membrane transporters. Conclusions The results from this metagenomic survey demonstrated the presence of genes associated with resistance to antibiotics and carbohydrate metabolism suggesting that the swine gut microbiome may be shaped by husbandry practices.

A metagenomic snapshot of taxonomic and functional diversity in an alpine glacier cryoconite ecosystem

International Nuclear Information System (INIS)

Edwards, Arwyn; Pachebat, Justin A; Swain, Martin; Hegarty, Matt; Rassner, Sara M E; Hodson, Andrew J; Irvine-Fynn, Tristram D L; Sattler, Birgit

2013-01-01

Cryoconite is a microbe–mineral aggregate which darkens the ice surface of glaciers. Microbial process and marker gene PCR-dependent measurements reveal active and diverse cryoconite microbial communities on polar glaciers. Here, we provide the first report of a cryoconite metagenome and culture-independent study of alpine cryoconite microbial diversity. We assembled 1.2 Gbp of metagenomic DNA sequenced using an Illumina HiScanSQ from cryoconite holes across the ablation zone of Rotmoosferner in the Austrian Alps. The metagenome revealed a bacterially-dominated community, with Proteobacteria (62% of bacterial-assigned contigs) and Bacteroidetes (14%) considerably more abundant than Cyanobacteria (2.5%). Streptophyte DNA dominated the eukaryotic metagenome. Functional genes linked to N, Fe, S and P cycling illustrated an acquisitive trend and a nitrogen cycle based upon efficient ammonia recycling. A comparison of 32 metagenome datasets revealed a similarity in functional profiles between the cryoconite and metagenomes characterized from other cold microbe–mineral aggregates. Overall, the metagenomic snapshot reveals the cryoconite ecosystem of this alpine glacier as dependent on scavenging carbon and nutrients from allochthonous sources, in particular mosses transported by wind from ice-marginal habitats, consistent with net heterotrophy indicated by productivity measurements. A transition from singular snapshots of cryoconite metagenomes to comparative analyses is advocated. (letter)

The Emerging Importance of IgG Fab Glycosylation in Immunity.

Science.gov (United States)

van de Bovenkamp, Fleur S; Hafkenscheid, Lise; Rispens, Theo; Rombouts, Yoann

2016-02-15

Human IgG is the most abundant glycoprotein in serum and is crucial for protective immunity. In addition to conserved IgG Fc glycans, ∼15-25% of serum IgG contains glycans within the variable domains. These so-called "Fab glycans" are primarily highly processed complex-type biantennary N-glycans linked to N-glycosylation sites that emerge during somatic hypermutation. Specific patterns of Fab glycosylation are concurrent with physiological and pathological conditions, such as pregnancy and rheumatoid arthritis. With respect to function, Fab glycosylation can significantly affect stability, half-life, and binding characteristics of Abs and BCRs. Moreover, Fab glycans are associated with the anti-inflammatory activity of IVIgs. Consequently, IgG Fab glycosylation appears to be an important, yet poorly understood, process that modulates immunity. Copyright © 2016 by The American Association of Immunologists, Inc.

In-depth resistome analysis by targeted metagenomics.

Science.gov (United States)

Lanza, Val F; Baquero, Fernando; Martínez, José Luís; Ramos-Ruíz, Ricardo; González-Zorn, Bruno; Andremont, Antoine; Sánchez-Valenzuela, Antonio; Ehrlich, Stanislav Dusko; Kennedy, Sean; Ruppé, Etienne; van Schaik, Willem; Willems, Rob J; de la Cruz, Fernando; Coque, Teresa M

2018-01-15

Antimicrobial resistance is a major global health challenge. Metagenomics allows analyzing the presence and dynamics of "resistomes" (the ensemble of genes encoding antimicrobial resistance in a given microbiome) in disparate microbial ecosystems. However, the low sensitivity and specificity of available metagenomic methods preclude the detection of minority populations (often present below their detection threshold) and/or the identification of allelic variants that differ in the resulting phenotype. Here, we describe a novel strategy that combines targeted metagenomics using last generation in-solution capture platforms, with novel bioinformatics tools to establish a standardized framework that allows both quantitative and qualitative analyses of resistomes. We developed ResCap, a targeted sequence capture platform based on SeqCapEZ (NimbleGene) technology, which includes probes for 8667 canonical resistance genes (7963 antibiotic resistance genes and 704 genes conferring resistance to metals or biocides), and 2517 relaxase genes (plasmid markers) and 78,600 genes homologous to the previous identified targets (47,806 for antibiotics and 30,794 for biocides or metals). Its performance was compared with metagenomic shotgun sequencing (MSS) for 17 fecal samples (9 humans, 8 swine). ResCap significantly improves MSS to detect "gene abundance" (from 2.0 to 83.2%) and "gene diversity" (26 versus 14.9 genes unequivocally detected per sample per million of reads; the number of reads unequivocally mapped increasing up to 300-fold by using ResCap), which were calculated using novel bioinformatic tools. ResCap also facilitated the analysis of novel genes potentially involved in the resistance to antibiotics, metals, biocides, or any combination thereof. ResCap, the first targeted sequence capture, specifically developed to analyze resistomes, greatly enhances the sensitivity and specificity of available metagenomic methods and offers the possibility to analyze genes

MetaVelvet: An Extension of Velvet Assembler to de novo Metagenome Assembly from Short Sequence Reads (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

Energy Technology Data Exchange (ETDEWEB)

Sakakibara, Yasumbumi

2011-10-13

Keio University's Yasumbumi Sakakibara on "MetaVelvet: An Extension of Velvet Assembler to de novo Metagenome Assembly from Short Sequence Reads" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

The potential of viral metagenomics in blood transfusion safety.

Science.gov (United States)

Sauvage, V; Gomez, J; Boizeau, L; Laperche, S

2017-09-01

Thanks to the significant advent of high throughput sequencing in the last ten years, it is now possible via metagenomics to define the spectrum of the microbial sequences present in human blood samples. Therefore, metagenomics sequencing appears as a promising approach for the identification and global surveillance of new, emerging and/or unexpected viruses that could impair blood transfusion safety. However, despite considerable advantages compared to the traditional methods of pathogen identification, this non-targeted approach presents several drawbacks including a lack of sensitivity and sequence contaminant issues. With further improvements, especially to increase sensitivity, metagenomics sequencing should become in a near future an additional diagnostic tool in infectious disease field and especially in blood transfusion safety. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Deciphering a pathway of Halobacterium salinarum N-glycosylation

Science.gov (United States)

Kandiba, Lina; Eichler, Jerry

2015-01-01

Genomic analysis points to N-glycosylation as being a common posttranslational modification in Archaea. To date, however, pathways of archaeal N-glycosylation have only been described for few species. With this in mind, the similarities of N-linked glycans decorating glycoproteins in the haloarchaea Haloferax volcanii and Halobacterium salinarum directed a series of bioinformatics, genetic, and biochemical experiments designed to describe that Hbt. salinarum pathway responsible for biogenesis of one of the two N-linked oligosaccharides described in this species. As in Hfx. volcanii, where agl (archaeal glycosylation) genes that encode proteins responsible for the assembly and attachment of a pentasaccharide to target protein Asn residues are clustered in the genome, Hbt. salinarum also contains a group of clustered homologous genes (VNG1048G-VNG1068G). Introduction of these Hbt. salinarum genes into Hfx. volcanii mutant strains deleted of the homologous sequence restored the lost activity. Moreover, transcription of the Hbt. salinarum genes in the native host, as well as in vitro biochemical confirmation of the predicted functions of several of the products of these genes provided further support for assignments made following bioinformatics and genetic experiments. Based on the results obtained in this study, the first description of an N-glycosylation pathway in Hbt. salinarum is offered. PMID:25461760

Challenges and Opportunities of Airborne Metagenomics

KAUST Repository

Behzad, H.; Gojobori, Takashi; Mineta, K.

2015-01-01

microorganisms. Airborne metagenomic studies could also lead to discoveries of novel genes and metabolic pathways relevant to meteorological and industrial applications, environmental bioremediation, and biogeochemical cycles.

MG-Digger: an automated pipeline to search for giant virus-related sequences in metagenomes

Directory of Open Access Journals (Sweden)

Jonathan eVerneau

2016-03-01

Full Text Available The number of metagenomic studies conducted each year is growing dramatically. Storage and analysis of such big data is difficult and time-consuming. Interestingly, analysis shows that environmental and human metagenomes include a significant amount of non-annotated sequences, representing a ‘dark matter’. We established a bioinformatics pipeline that automatically detects metagenome reads matching query sequences from a given set and applied this tool to the detection of sequences matching large and giant DNA viral members of the proposed order Megavirales or virophages. A total of 1,045 environmental and human metagenomes (≈ 1 Terabase pairs were collected, processed and stored on our bioinformatics server. In addition, nucleotide and protein sequences from 93 Megavirales representatives, including 19 giant viruses of amoeba, and five virophages, were collected. The pipeline was generated by scripts written in Python language and entitled MG-Digger. Metagenomes previously found to contain megavirus-like sequences were tested as controls. MG-Digger was able to annotate hundreds of metagenome sequences as best matching those of giant viruses. These sequences were most often found to be similar to phycodnavirus or mimivirus sequences, but included reads related to recently available pandoraviruses, Pithovirus sibericum, and faustoviruses. Compared to other tools, MG-Digger combined stand-alone use on Linux or Windows operating systems through a user-friendly interface, implementation of ready-to-use customized metagenome databases and query sequence databases, adjustable parameters for BLAST searches, and creation of output files containing selected reads with best match identification. Compared to Metavir 2, a reference tool in viral metagenome analysis, MG-Digger detected 8% more true positive Megavirales-related reads in a control metagenome. The present work shows that massive, automated and recurrent analyses of metagenomes are

Neuronal glycosylation differentials in normal, injured and chondroitinase-treated environments

International Nuclear Information System (INIS)

Kilcoyne, Michelle; Sharma, Shashank; McDevitt, Niamh; O’Leary, Claire; Joshi, Lokesh; McMahon, Siobhán S.

2012-01-01

Highlights: ► Carbohydrates are important in the CNS and ChABC has been used for spinal cord injury (SCI) treatment. ► Neuronal glycosylation in injury and after ChABC treatment is unknown. ► In silico mining verified that glyco-related genes were differentially regulated after SCI. ► In vitro model system revealed abnormal sialylation in an injured environment. ► The model indicated a return to normal neuronal glycosylation after ChABC treatment. -- Abstract: Glycosylation is found ubiquitously throughout the central nervous system (CNS). Chondroitin sulphate proteoglycans (CSPGs) are a group of molecules heavily substituted with glycosaminoglycans (GAGs) and are found in the extracellular matrix (ECM) and cell surfaces. Upon CNS injury, a glial scar is formed, which is inhibitory for axon regeneration. Several CSPGs are up-regulated within the glial scar, including NG2, and these CSPGs are key inhibitory molecules of axonal regeneration. Treatment with chondroitinase ABC (ChABC) can neutralise the inhibitory nature of NG2. A gene expression dataset was mined in silico to verify differentially regulated glycosylation-related genes in neurons after spinal cord injury and identify potential targets for further investigation. To establish the glycosylation differential of neurons that grow in a healthy, inhibitory and ChABC-treated environment, we established an indirect co-culture system where PC12 neurons were grown with primary astrocytes, Neu7 astrocytes (which overexpress NG2) and Neu7 astrocytes treated with ChABC. After 1, 4 and 8 days culture, lectin cytochemistry of the neurons was performed using five fluorescently-labelled lectins (ECA MAA, PNA, SNA-I and WFA). Usually α-(2,6)-linked sialylation scarcely occurs in the CNS but this motif was observed on the neurons in the injured environment only at day 8. Treatment with ChABC was successful in returning neuronal glycosylation to normal conditions at all timepoints for MAA, PNA and SNA-I staining

Neuronal glycosylation differentials in normal, injured and chondroitinase-treated environments

Energy Technology Data Exchange (ETDEWEB)

Kilcoyne, Michelle; Sharma, Shashank [Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland, Galway (Ireland); McDevitt, Niamh; O' Leary, Claire [Anatomy, School of Medicine, National University of Ireland, Galway (Ireland); Joshi, Lokesh [Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland, Galway (Ireland); McMahon, Siobhan S., E-mail: siobhan.mcmahon@nuigalway.ie [Anatomy, School of Medicine, National University of Ireland, Galway (Ireland)

2012-04-13

Highlights: Black-Right-Pointing-Pointer Carbohydrates are important in the CNS and ChABC has been used for spinal cord injury (SCI) treatment. Black-Right-Pointing-Pointer Neuronal glycosylation in injury and after ChABC treatment is unknown. Black-Right-Pointing-Pointer In silico mining verified that glyco-related genes were differentially regulated after SCI. Black-Right-Pointing-Pointer In vitro model system revealed abnormal sialylation in an injured environment. Black-Right-Pointing-Pointer The model indicated a return to normal neuronal glycosylation after ChABC treatment. -- Abstract: Glycosylation is found ubiquitously throughout the central nervous system (CNS). Chondroitin sulphate proteoglycans (CSPGs) are a group of molecules heavily substituted with glycosaminoglycans (GAGs) and are found in the extracellular matrix (ECM) and cell surfaces. Upon CNS injury, a glial scar is formed, which is inhibitory for axon regeneration. Several CSPGs are up-regulated within the glial scar, including NG2, and these CSPGs are key inhibitory molecules of axonal regeneration. Treatment with chondroitinase ABC (ChABC) can neutralise the inhibitory nature of NG2. A gene expression dataset was mined in silico to verify differentially regulated glycosylation-related genes in neurons after spinal cord injury and identify potential targets for further investigation. To establish the glycosylation differential of neurons that grow in a healthy, inhibitory and ChABC-treated environment, we established an indirect co-culture system where PC12 neurons were grown with primary astrocytes, Neu7 astrocytes (which overexpress NG2) and Neu7 astrocytes treated with ChABC. After 1, 4 and 8 days culture, lectin cytochemistry of the neurons was performed using five fluorescently-labelled lectins (ECA MAA, PNA, SNA-I and WFA). Usually {alpha}-(2,6)-linked sialylation scarcely occurs in the CNS but this motif was observed on the neurons in the injured environment only at day 8. Treatment

DISAL glycosyl donors for the synthesis of a linear hexasaccharide under mild conditions

DEFF Research Database (Denmark)

Petersen, Lars; Laursen, Jane B.; Larsen, K.

2003-01-01

The new class of glycosyl donors with a methyl 3,5-dinitrosalicylate (DISAL) anomeric leaving group has proved efficient for glycosylation under strictly neutral, mildly basic, or mildly acidic conditions. Here, we report the synthesis of novel DISAL disaccharide glycosyl donors prepared by easy...... nucleophilic aromatic substitution. These DISAL donors proved efficient in the synthesis of a starch-related hexasaccharide under very mild conditions. Glycosylations proceeded with alpha-selectivity and were compatible with Trt protecting groups....

Integrative Workflows for Metagenomic Analysis

Directory of Open Access Journals (Sweden)

Efthymios eLadoukakis

2014-11-01

Full Text Available The rapid evolution of all sequencing technologies, described by the term Next Generation Sequencing (NGS, have revolutionized metagenomic analysis. They constitute a combination of high-throughput analytical protocols, coupled to delicate measuring techniques, in order to potentially discover, properly assemble and map allelic sequences to the correct genomes, achieving particularly high yields for only a fraction of the cost of traditional processes (i.e. Sanger. From a bioinformatic perspective, this boils down to many gigabytes of data being generated from each single sequencing experiment, rendering the management or even the storage, critical bottlenecks with respect to the overall analytical endeavor. The enormous complexity is even more aggravated by the versatility of the processing steps available, represented by the numerous bioinformatic tools that are essential, for each analytical task, in order to fully unveil the genetic content of a metagenomic dataset. These disparate tasks range from simple, nonetheless non-trivial, quality control of raw data to exceptionally complex protein annotation procedures, requesting a high level of expertise for their proper application or the neat implementation of the whole workflow. Furthermore, a bioinformatic analysis of such scale, requires grand computational resources, imposing as the sole realistic solution, the utilization of cloud computing infrastructures. In this review article we discuss different, integrative, bioinformatic solutions available, which address the aforementioned issues, by performing a critical assessment of the available automated pipelines for data management, quality control and annotation of metagenomic data, embracing various, major sequencing technologies and applications.

Flagellar glycosylation in Clostridium botulinum.

Science.gov (United States)

Twine, Susan M; Paul, Catherine J; Vinogradov, Evgeny; McNally, David J; Brisson, Jean-Robert; Mullen, James A; McMullin, David R; Jarrell, Harold C; Austin, John W; Kelly, John F; Logan, Susan M

2008-09-01

Flagellins from Clostridium botulinum were shown to be post-translationally modified with novel glycan moieties by top-down MS analysis of purified flagellin protein from strains of various toxin serotypes. Detailed analyses of flagellin from two strains of C. botulinum demonstrated that the protein is modified by a novel glycan moiety of mass 417 Da in O-linkage. Bioinformatic analysis of available C. botulinum genomes identified a flagellar glycosylation island containing homologs of genes recently identified in Campylobacter coli that have been shown to be responsible for the biosynthesis of legionaminic acid derivatives. Structural characterization of the carbohydrate moiety was completed utilizing both MS and NMR spectroscopy, and it was shown to be a novel legionaminic acid derivative, 7-acetamido-5-(N-methyl-glutam-4-yl)-amino-3,5,7,9-tetradeoxy-D-glycero-alpha-D-galacto-nonulosonic acid, (alphaLeg5GluNMe7Ac). Electron transfer dissociation MS with and without collision-activated dissociation was utilized to map seven sites of O-linked glycosylation, eliminating the need for chemical derivatization of tryptic peptides prior to analysis. Marker ions for novel glycans, as well as a unique C-terminal flagellin peptide marker ion, were identified in a top-down analysis of the intact protein. These ions have the potential for use in for rapid detection and discrimination of C. botulinum cells, indicating botulinum neurotoxin contamination. This is the first report of glycosylation of Gram-positive flagellar proteins by the 'sialic acid-like' nonulosonate sugar, legionaminic acid.

Metaviz: interactive statistical and visual analysis of metagenomic data.

Science.gov (United States)

Wagner, Justin; Chelaru, Florin; Kancherla, Jayaram; Paulson, Joseph N; Zhang, Alexander; Felix, Victor; Mahurkar, Anup; Elmqvist, Niklas; Corrada Bravo, Héctor

2018-04-06

Large studies profiling microbial communities and their association with healthy or disease phenotypes are now commonplace. Processed data from many of these studies are publicly available but significant effort is required for users to effectively organize, explore and integrate it, limiting the utility of these rich data resources. Effective integrative and interactive visual and statistical tools to analyze many metagenomic samples can greatly increase the value of these data for researchers. We present Metaviz, a tool for interactive exploratory data analysis of annotated microbiome taxonomic community profiles derived from marker gene or whole metagenome shotgun sequencing. Metaviz is uniquely designed to address the challenge of browsing the hierarchical structure of metagenomic data features while rendering visualizations of data values that are dynamically updated in response to user navigation. We use Metaviz to provide the UMD Metagenome Browser web service, allowing users to browse and explore data for more than 7000 microbiomes from published studies. Users can also deploy Metaviz as a web service, or use it to analyze data through the metavizr package to interoperate with state-of-the-art analysis tools available through Bioconductor. Metaviz is free and open source with the code, documentation and tutorials publicly accessible.

Diversity Indices as Measures of Functional Annotation Methods in Metagenomics Studies

KAUST Repository

Jankovic, Boris R.

2016-01-26

Applications of high-throughput techniques in metagenomics studies produce massive amounts of data. Fragments of genomic, transcriptomic and proteomic molecules are all found in metagenomics samples. Laborious and meticulous effort in sequencing and functional annotation are then required to, amongst other objectives, reconstruct a taxonomic map of the environment that metagenomics samples were taken from. In addition to computational challenges faced by metagenomics studies, the analysis is further complicated by the presence of contaminants in the samples, potentially resulting in skewed taxonomic analysis. The functional annotation in metagenomics can utilize all available omics data and therefore different methods that are associated with a particular type of data. For example, protein-coding DNA, non-coding RNA or ribosomal RNA data can be used in such an analysis. These methods would have their advantages and disadvantages and the question of comparison among them naturally arises. There are several criteria that can be used when performing such a comparison. Loosely speaking, methods can be evaluated in terms of computational complexity or in terms of the expected biological accuracy. We propose that the concept of diversity that is used in the ecosystems and species diversity studies can be successfully used in evaluating certain aspects of the methods employed in metagenomics studies. We show that when applying the concept of Hill’s diversity, the analysis of variations in the diversity order provides valuable clues into the robustness of methods used in the taxonomical analysis.

Phylogenetic convolutional neural networks in metagenomics.

Science.gov (United States)

Fioravanti, Diego; Giarratano, Ylenia; Maggio, Valerio; Agostinelli, Claudio; Chierici, Marco; Jurman, Giuseppe; Furlanello, Cesare

2018-03-08

Convolutional Neural Networks can be effectively used only when data are endowed with an intrinsic concept of neighbourhood in the input space, as is the case of pixels in images. We introduce here Ph-CNN, a novel deep learning architecture for the classification of metagenomics data based on the Convolutional Neural Networks, with the patristic distance defined on the phylogenetic tree being used as the proximity measure. The patristic distance between variables is used together with a sparsified version of MultiDimensional Scaling to embed the phylogenetic tree in a Euclidean space. Ph-CNN is tested with a domain adaptation approach on synthetic data and on a metagenomics collection of gut microbiota of 38 healthy subjects and 222 Inflammatory Bowel Disease patients, divided in 6 subclasses. Classification performance is promising when compared to classical algorithms like Support Vector Machines and Random Forest and a baseline fully connected neural network, e.g. the Multi-Layer Perceptron. Ph-CNN represents a novel deep learning approach for the classification of metagenomics data. Operatively, the algorithm has been implemented as a custom Keras layer taking care of passing to the following convolutional layer not only the data but also the ranked list of neighbourhood of each sample, thus mimicking the case of image data, transparently to the user.

Metagenomic sequence of saline desert microbiota from wild ass sanctuary, Little Rann of Kutch, Gujarat, India.

Science.gov (United States)

Patel, Rajesh; Mevada, Vishal; Prajapati, Dhaval; Dudhagara, Pravin; Koringa, Prakash; Joshi, C G

2015-03-01

We report Metagenome from the saline desert soil sample of Little Rann of Kutch, Gujarat State, India. Metagenome consisted of 633,760 sequences with size 141,307,202 bp and 56% G + C content. Metagenome sequence data are available at EBI under EBI Metagenomics database with accession no. ERP005612. Community metagenomics revealed total 1802 species belonged to 43 different phyla with dominating Marinobacter (48.7%) and Halobacterium (4.6%) genus in bacterial and archaeal domain respectively. Remarkably, 18.2% sequences in a poorly characterized group and 4% gene for various stress responses along with versatile presence of commercial enzyme were evident in a functional metagenome analysis.

Glycogenomics as a mass spectrometry-guided genome-mining method for microbial glycosylated molecules.

Science.gov (United States)

Kersten, Roland D; Ziemert, Nadine; Gonzalez, David J; Duggan, Brendan M; Nizet, Victor; Dorrestein, Pieter C; Moore, Bradley S

2013-11-19

Glycosyl groups are an essential mediator of molecular interactions in cells and on cellular surfaces. There are very few methods that directly relate sugar-containing molecules to their biosynthetic machineries. Here, we introduce glycogenomics as an experiment-guided genome-mining approach for fast characterization of glycosylated natural products (GNPs) and their biosynthetic pathways from genome-sequenced microbes by targeting glycosyl groups in microbial metabolomes. Microbial GNPs consist of aglycone and glycosyl structure groups in which the sugar unit(s) are often critical for the GNP's bioactivity, e.g., by promoting binding to a target biomolecule. GNPs are a structurally diverse class of molecules with important pharmaceutical and agrochemical applications. Herein, O- and N-glycosyl groups are characterized in their sugar monomers by tandem mass spectrometry (MS) and matched to corresponding glycosylation genes in secondary metabolic pathways by a MS-glycogenetic code. The associated aglycone biosynthetic genes of the GNP genotype then classify the natural product to further guide structure elucidation. We highlight the glycogenomic strategy by the characterization of several bioactive glycosylated molecules and their gene clusters, including the anticancer agent cinerubin B from Streptomyces sp. SPB74 and an antibiotic, arenimycin B, from Salinispora arenicola CNB-527.

Metagenomic Taxonomy-Guided Database-Searching Strategy for Improving Metaproteomic Analysis.

Science.gov (United States)

Xiao, Jinqiu; Tanca, Alessandro; Jia, Ben; Yang, Runqing; Wang, Bo; Zhang, Yu; Li, Jing

2018-04-06

Metaproteomics provides a direct measure of the functional information by investigating all proteins expressed by a microbiota. However, due to the complexity and heterogeneity of microbial communities, it is very hard to construct a sequence database suitable for a metaproteomic study. Using a public database, researchers might not be able to identify proteins from poorly characterized microbial species, while a sequencing-based metagenomic database may not provide adequate coverage for all potentially expressed protein sequences. To address this challenge, we propose a metagenomic taxonomy-guided database-search strategy (MT), in which a merged database is employed, consisting of both taxonomy-guided reference protein sequences from public databases and proteins from metagenome assembly. By applying our MT strategy to a mock microbial mixture, about two times as many peptides were detected as with the metagenomic database only. According to the evaluation of the reliability of taxonomic attribution, the rate of misassignments was comparable to that obtained using an a priori matched database. We also evaluated the MT strategy with a human gut microbial sample, and we found 1.7 times as many peptides as using a standard metagenomic database. In conclusion, our MT strategy allows the construction of databases able to provide high sensitivity and precision in peptide identification in metaproteomic studies, enabling the detection of proteins from poorly characterized species within the microbiota.

Nonenzymatic glycosylation of bovine myelin basic protein

International Nuclear Information System (INIS)

Hitz, J.B.

1987-01-01

In the CNS myelin sheath the nonenzymatic glycosylation reaction (at the early stage of the Amadori product) occurs only with the myelin basic protein and not with the other myelin proteins. This was observed in isolated bovine myelin by in vitro incubation with [ 14 C]-galactose and [ 14 C]-glucose. The respective in-vitro incorporation rates for purified bovine myelin basic protein with D-galactose, D-glucose and D-mannose were 7.2, 2.4 and 2.4 mmoles/mole myelin basic protein per day at 37 0 C. A more rapid, HPLC method was devised and characterized to specifically analyze for the Amadori product. The HPLC method was correlated to the [ 14 C]-sugar incorporation method for myelin basic protein under a set of standard reaction conditions using [ 14 C]-glucose and [ 14 C]-mannose with HPLC values at 1/6 and 1/5 of the [ 14 C]-sugar incorporation method. A novel myelin basic protein purification step has been developed that yields a relativity proteolytic free preparation that is easy to work with, being totally soluble at a neutral pH. Nine new spots appear for a trypsinized glycosylated MBP in the paper peptide map of which eight correspond to positions of the [ 3 H]-labeled Amadori product in affinity isolated peptides. These studies provide a general characterization of and a structural basis for investigations on nonenzymatically glycosylated MBP as well as identifying MBP as the only nonenzymatically glycosylated protein in the CNS myelin sheath which may accumulate during aging, diabetes, and demyelinating diseases in general

Evaluation of the Cow Rumen Metagenome: Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

Energy Technology Data Exchange (ETDEWEB)

Sczyrba, Alex

2011-10-13

DOE JGI's Alex Sczyrba on "Evaluation of the Cow Rumen Metagenome" and "Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

The effect of glycosylation on cytotoxicity of Ibaraki virus nonstructural protein NS3

Science.gov (United States)

URATA, Maho; WATANABE, Rie; IWATA, Hiroyuki

2015-01-01

The cytotoxicity of Ibaraki virus nonstructural protein NS3 was confirmed, and the contribution of glycosylation to this activity was examined by using glycosylation mutants of NS3 generated by site-directed mutagenesis. The expression of NS3 resulted in leakage of lactate dehydrogenase to the culture supernatant, suggesting the cytotoxicity of this protein. The lack of glycosylation impaired the transport of NS3 to the plasma membrane and resulted in reduced cytotoxicity. Combined with the previous observation that NS3 glycosylation was specifically observed in mammalian cells (Urata et al., Virus Research 2014), it was suggested that the alteration of NS3 cytotoxicity through modulating glycosylation is one of the strategies to achieve host specific pathogenisity of Ibaraki virus between mammals and vector arthropods. PMID:26178820

Reconstruction of ribosomal RNA genes from metagenomic data.

Directory of Open Access Journals (Sweden)

Lu Fan

Full Text Available Direct sequencing of environmental DNA (metagenomics has a great potential for describing the 16S rRNA gene diversity of microbial communities. However current approaches using this 16S rRNA gene information to describe community diversity suffer from low taxonomic resolution or chimera problems. Here we describe a new strategy that involves stringent assembly and data filtering to reconstruct full-length 16S rRNA genes from metagenomicpyrosequencing data. Simulations showed that reconstructed 16S rRNA genes provided a true picture of the community diversity, had minimal rates of chimera formation and gave taxonomic resolution down to genus level. The strategy was furthermore compared to PCR-based methods to determine the microbial diversity in two marine sponges. This showed that about 30% of the abundant phylotypes reconstructed from metagenomic data failed to be amplified by PCR. Our approach is readily applicable to existing metagenomic datasets and is expected to lead to the discovery of new microbial phylotypes.

Bioinformatics tools for quantitative and functional metagenome and metatranscriptome data analysis in microbes.

Science.gov (United States)

Niu, Sheng-Yong; Yang, Jinyu; McDermaid, Adam; Zhao, Jing; Kang, Yu; Ma, Qin

2017-05-08

Metagenomic and metatranscriptomic sequencing approaches are more frequently being used to link microbiota to important diseases and ecological changes. Many analyses have been used to compare the taxonomic and functional profiles of microbiota across habitats or individuals. While a large portion of metagenomic analyses focus on species-level profiling, some studies use strain-level metagenomic analyses to investigate the relationship between specific strains and certain circumstances. Metatranscriptomic analysis provides another important insight into activities of genes by examining gene expression levels of microbiota. Hence, combining metagenomic and metatranscriptomic analyses will help understand the activity or enrichment of a given gene set, such as drug-resistant genes among microbiome samples. Here, we summarize existing bioinformatics tools of metagenomic and metatranscriptomic data analysis, the purpose of which is to assist researchers in deciding the appropriate tools for their microbiome studies. Additionally, we propose an Integrated Meta-Function mapping pipeline to incorporate various reference databases and accelerate functional gene mapping procedures for both metagenomic and metatranscriptomic analyses. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

N-Glycosylation of Carnosinase Influences Protein Secretion and Enzyme Activity Implications for Hyperglycemia

NARCIS (Netherlands)

Riedl, Eva; Koeppel, Hannes; Pfister, Frederick; Peters, Verena; Sauerhoefer, Sibylle; Sternik, Paula; Brinkkoetter, Paul; Zentgraf, Hanswalter; Navis, Gerjan; Henning, Robert H.; Van Den Born, Jacob; Bakker, Stephan J. L.; Janssen, Bart; van der Woude, Fokko J.; Yard, Benito A.

OBJECTIVE-The (CTG)(n) polymorphism in the serum carnosinase (CN-1) gene affects CN-1 secretion Since CN-1 is heavily glycosylated and glycosylation might influence protein secretion as well, we tested the role of N-glycosylation for CN-1 secretion and enzyme activity. We also tested whether CN-1

Exploration of Metagenome Assemblies with an Interactive Visualization Tool

Energy Technology Data Exchange (ETDEWEB)

Cantor, Michael; Nordberg, Henrik; Smirnova, Tatyana; Andersen, Evan; Tringe, Susannah; Hess, Matthias; Dubchak, Inna

2014-07-09

Metagenomics, one of the fastest growing areas of modern genomic science, is the genetic profiling of the entire community of microbial organisms present in an environmental sample. Elviz is a web-based tool for the interactive exploration of metagenome assemblies. Elviz can be used with publicly available data sets from the Joint Genome Institute or with custom user-loaded assemblies. Elviz is available at genome.jgi.doe.gov/viz

Cancer associated aberrant protein o-glycosylation can modify antigen processing and immune response

DEFF Research Database (Denmark)

Madsen, Caroline B; Petersen, Cecilie; Lavrsen, Kirstine

2012-01-01

Aberrant glycosylation of mucins and other extracellular proteins is an important event in carcinogenesis and the resulting cancer associated glycans have been suggested as targets in cancer immunotherapy. We assessed the role of O-linked GalNAc glycosylation on antigen uptake, processing......, and presentation on MHC class I and II molecules. The effect of GalNAc O-glycosylation was monitored with a model system based on ovalbumin (OVA)-MUC1 fusion peptides (+/- glycosylation) loaded onto dendritic cells co-cultured with IL-2 secreting OVA peptide-specific T cell hybridomas. To evaluate the in vivo...

Rapid phenolic O-glycosylation of small molecules and complex unprotected peptides in aqueous solvent

Science.gov (United States)

Wadzinski, Tyler J.; Steinauer, Angela; Hie, Liana; Pelletier, Guillaume; Schepartz, Alanna; Miller, Scott J.

2018-06-01

Glycosylated natural products and synthetic glycopeptides represent a significant and growing source of biochemical probes and therapeutic agents. However, methods that enable the aqueous glycosylation of endogenous amino acid functionality in peptides without the use of protecting groups are scarce. Here, we report a transformation that facilitates the efficient aqueous O-glycosylation of phenolic functionality in a wide range of small molecules, unprotected tyrosine, and tyrosine residues embedded within a range of complex, fully unprotected peptides. The transformation, which uses glycosyl fluoride donors and is promoted by Ca(OH)2, proceeds rapidly at room temperature in water, with good yields and selective formation of unique anomeric products depending on the stereochemistry of the glycosyl donor. High functional group tolerance is observed, and the phenol glycosylation occurs selectively in the presence of virtually all side chains of the proteinogenic amino acids with the singular exception of Cys. This method offers a highly selective, efficient, and operationally simple approach for the protecting-group-free synthesis of O-aryl glycosides and Tyr-O-glycosylated peptides in water.

Metagenomic frameworks for monitoring antibiotic resistance in aquatic environments.

Science.gov (United States)

Port, Jesse A; Cullen, Alison C; Wallace, James C; Smith, Marissa N; Faustman, Elaine M

2014-03-01

High-throughput genomic technologies offer new approaches for environmental health monitoring, including metagenomic surveillance of antibiotic resistance determinants (ARDs). Although natural environments serve as reservoirs for antibiotic resistance genes that can be transferred to pathogenic and human commensal bacteria, monitoring of these determinants has been infrequent and incomplete. Furthermore, surveillance efforts have not been integrated into public health decision making. We used a metagenomic epidemiology-based approach to develop an ARD index that quantifies antibiotic resistance potential, and we analyzed this index for common modal patterns across environmental samples. We also explored how metagenomic data such as this index could be conceptually framed within an early risk management context. We analyzed 25 published data sets from shotgun pyrosequencing projects. The samples consisted of microbial community DNA collected from marine and freshwater environments across a gradient of human impact. We used principal component analysis to identify index patterns across samples. We observed significant differences in the overall index and index subcategory levels when comparing ecosystems more proximal versus distal to human impact. The selection of different sequence similarity thresholds strongly influenced the index measurements. Unique index subcategory modes distinguished the different metagenomes. Broad-scale screening of ARD potential using this index revealed utility for framing environmental health monitoring and surveillance. This approach holds promise as a screening tool for establishing baseline ARD levels that can be used to inform and prioritize decision making regarding management of ARD sources and human exposure routes. Port JA, Cullen AC, Wallace JC, Smith MN, Faustman EM. 2014. Metagenomic frameworks for monitoring antibiotic resistance in aquatic environments. Environ Health Perspect 122:222–228; http://dx.doi.org/10.1289/ehp

[Conformation analysis of the N-glycosylation site Asn-X-Thr/Ser in glycoproteins].

Science.gov (United States)

Avanov, A Ia; Lipkind, G M

1990-03-01

Theoretical conformational analysis of oligopeptides CH3CO-Asn-X-Thr-NHCH3 (X = Gly, Ala, Pro), modelling N-glycosylation site, and their glycosylated derivatives CH3CO-(GlcNAc beta 1-4GlcNAc beta 1) Asn-X-Thr-NHCH3 has been carried out. Active conformations of the site are found, corresponding to structural prerequisities of N-glycosylation: Asn residue's position in beta-turn and hydrogen bond formation between side chains of Asn and Thr/Ser residues. In this case the L conformation of the central residue X is most probable. Since Pro residue does not possess this conformation, sequences with X = Pro are not glycosylated. It is shown that glycosylation of the above-mentioned sites is accompanied by reorientation of the Asn residue's side chains.

A computational framework for the automated construction of glycosylation reaction networks.

Science.gov (United States)

Liu, Gang; Neelamegham, Sriram

2014-01-01

Glycosylation is among the most common and complex post-translational modifications identified to date. It proceeds through the catalytic action of multiple enzyme families that include the glycosyltransferases that add monosaccharides to growing glycans, and glycosidases which remove sugar residues to trim glycans. The expression level and specificity of these enzymes, in part, regulate the glycan distribution or glycome of specific cell/tissue systems. Currently, there is no systematic method to describe the enzymes and cellular reaction networks that catalyze glycosylation. To address this limitation, we present a streamlined machine-readable definition for the glycosylating enzymes and additional methodologies to construct and analyze glycosylation reaction networks. In this computational framework, the enzyme class is systematically designed to store detailed specificity data such as enzymatic functional group, linkage and substrate specificity. The new classes and their associated functions enable both single-reaction inference and automated full network reconstruction, when given a list of reactants and/or products along with the enzymes present in the system. In addition, graph theory is used to support functions that map the connectivity between two or more species in a network, and that generate subset models to identify rate-limiting steps regulating glycan biosynthesis. Finally, this framework allows the synthesis of biochemical reaction networks using mass spectrometry (MS) data. The features described above are illustrated using three case studies that examine: i) O-linked glycan biosynthesis during the construction of functional selectin-ligands; ii) automated N-linked glycosylation pathway construction; and iii) the handling and analysis of glycomics based MS data. Overall, the new computational framework enables automated glycosylation network model construction and analysis by integrating knowledge of glycan structure and enzyme biochemistry. All

A computational framework for the automated construction of glycosylation reaction networks.

Directory of Open Access Journals (Sweden)

Gang Liu

Full Text Available Glycosylation is among the most common and complex post-translational modifications identified to date. It proceeds through the catalytic action of multiple enzyme families that include the glycosyltransferases that add monosaccharides to growing glycans, and glycosidases which remove sugar residues to trim glycans. The expression level and specificity of these enzymes, in part, regulate the glycan distribution or glycome of specific cell/tissue systems. Currently, there is no systematic method to describe the enzymes and cellular reaction networks that catalyze glycosylation. To address this limitation, we present a streamlined machine-readable definition for the glycosylating enzymes and additional methodologies to construct and analyze glycosylation reaction networks. In this computational framework, the enzyme class is systematically designed to store detailed specificity data such as enzymatic functional group, linkage and substrate specificity. The new classes and their associated functions enable both single-reaction inference and automated full network reconstruction, when given a list of reactants and/or products along with the enzymes present in the system. In addition, graph theory is used to support functions that map the connectivity between two or more species in a network, and that generate subset models to identify rate-limiting steps regulating glycan biosynthesis. Finally, this framework allows the synthesis of biochemical reaction networks using mass spectrometry (MS data. The features described above are illustrated using three case studies that examine: i O-linked glycan biosynthesis during the construction of functional selectin-ligands; ii automated N-linked glycosylation pathway construction; and iii the handling and analysis of glycomics based MS data. Overall, the new computational framework enables automated glycosylation network model construction and analysis by integrating knowledge of glycan structure and enzyme

Comprehensive benchmarking and ensemble approaches for metagenomic classifiers.

Science.gov (United States)

McIntyre, Alexa B R; Ounit, Rachid; Afshinnekoo, Ebrahim; Prill, Robert J; Hénaff, Elizabeth; Alexander, Noah; Minot, Samuel S; Danko, David; Foox, Jonathan; Ahsanuddin, Sofia; Tighe, Scott; Hasan, Nur A; Subramanian, Poorani; Moffat, Kelly; Levy, Shawn; Lonardi, Stefano; Greenfield, Nick; Colwell, Rita R; Rosen, Gail L; Mason, Christopher E

2017-09-21

One of the main challenges in metagenomics is the identification of microorganisms in clinical and environmental samples. While an extensive and heterogeneous set of computational tools is available to classify microorganisms using whole-genome shotgun sequencing data, comprehensive comparisons of these methods are limited. In this study, we use the largest-to-date set of laboratory-generated and simulated controls across 846 species to evaluate the performance of 11 metagenomic classifiers. Tools were characterized on the basis of their ability to identify taxa at the genus, species, and strain levels, quantify relative abundances of taxa, and classify individual reads to the species level. Strikingly, the number of species identified by the 11 tools can differ by over three orders of magnitude on the same datasets. Various strategies can ameliorate taxonomic misclassification, including abundance filtering, ensemble approaches, and tool intersection. Nevertheless, these strategies were often insufficient to completely eliminate false positives from environmental samples, which are especially important where they concern medically relevant species. Overall, pairing tools with different classification strategies (k-mer, alignment, marker) can combine their respective advantages. This study provides positive and negative controls, titrated standards, and a guide for selecting tools for metagenomic analyses by comparing ranges of precision, accuracy, and recall. We show that proper experimental design and analysis parameters can reduce false positives, provide greater resolution of species in complex metagenomic samples, and improve the interpretation of results.

Cell Surface Glycosylation Is Required for Efficient Mating of Haloferax volcanii

Directory of Open Access Journals (Sweden)

Yarden Shalev

2017-07-01

Full Text Available Halophilic archaea use a fusion-based mating system for lateral gene transfer across cells, yet the molecular mechanisms involved remain unknown. Previous work implied that cell fusion involves cell–cell recognition since fusion occurs more efficiently between cells from the same species. Long believed to be restricted only to Eukarya, it is now known that cells of all three domains of life perform N-glycosylation, the covalent attachment of glycans to select target asparagine residues in proteins, and that this post-translational modification is common for archaeal cell surface proteins. Here, we show that differences in glycosylation of the Haloferax volcanii surface-layer glycoprotein, brought about either by changing medium salinity or by knocking out key glycosylation genes, reduced mating success. Thus, different glycosylation patterns are likely to underlie mating preference in halophilic archaea, contributing to speciation processes.

Metagenomic Analysis of Dairy Bacteriophages

DEFF Research Database (Denmark)

Muhammed, Musemma K.; Kot, Witold; Neve, Horst

2017-01-01

Despite their huge potential for characterizing the biodiversity of phages, metagenomic studies are currently not available for dairy bacteriophages, partly due to the lack of a standard procedure for phage extraction. We optimized an extraction method that allows to remove the bulk protein from...

ELIXIR pilot action: Marine metagenomics - towards a domain specific set of sustainable services.

Science.gov (United States)

Robertsen, Espen Mikal; Denise, Hubert; Mitchell, Alex; Finn, Robert D; Bongo, Lars Ailo; Willassen, Nils Peder

2017-01-01

Metagenomics, the study of genetic material recovered directly from environmental samples, has the potential to provide insight into the structure and function of heterogeneous microbial communities.  There has been an increased use of metagenomics to discover and understand the diverse biosynthetic capacities of marine microbes, thereby allowing them to be exploited for industrial, food, and health care products. This ELIXIR pilot action was motivated by the need to establish dedicated data resources and harmonized metagenomics pipelines for the marine domain, in order to enhance the exploration and exploitation of marine genetic resources. In this paper, we summarize some of the results from the ELIXIR pilot action "Marine metagenomics - towards user centric services".

A Novel Strategy for Characterization of Glycosylated Proteins Separated by Gel Electrophoresis

DEFF Research Database (Denmark)

Larsen, Martin; Skottrup, Peter; Enghild, Jan Johannes

Protein glycosylation can be vital for changing the function or physiochemical properties of a protein. Abnormal glycosylation can lead to protein malfunction, resulting in severe diseases. Therefore, it is important to develop techniques for characterization of such modifications in proteins...... graphite powder micro-columns in combination with mass spectrometry. The method is faster and more sensitive than previous approaches and would be ideal for proteomics studies and verification of correct glycosylation of recombinant glycoproteins....

Challenges and opportunities in understanding microbial communities with metagenome assembly (accompanied by IPython Notebook tutorial)

Science.gov (United States)

Howe, Adina; Chain, Patrick S. G.

2015-01-01

Metagenomic investigations hold great promise for informing the genetics, physiology, and ecology of environmental microorganisms. Current challenges for metagenomic analysis are related to our ability to connect the dots between sequencing reads, their population of origin, and their encoding functions. Assembly-based methods reduce dataset size by extending overlapping reads into larger contiguous sequences (contigs), providing contextual information for genetic sequences that does not rely on existing references. These methods, however, tend to be computationally intensive and are again challenged by sequencing errors as well as by genomic repeats While numerous tools have been developed based on these methodological concepts, they present confounding choices and training requirements to metagenomic investigators. To help with accessibility to assembly tools, this review also includes an IPython Notebook metagenomic assembly tutorial. This tutorial has instructions for execution any operating system using Amazon Elastic Cloud Compute and guides users through downloading, assembly, and mapping reads to contigs of a mock microbiome metagenome. Despite its challenges, metagenomic analysis has already revealed novel insights into many environments on Earth. As software, training, and data continue to emerge, metagenomic data access and its discoveries will to grow. PMID:26217314

Challenges and opportunities in understanding microbial communities with metagenome assembly (accompanied by IPython Notebook tutorial

Directory of Open Access Journals (Sweden)

Adina eHowe

2015-07-01

Full Text Available Metagenomic investigations hold great promise for informing the genetics, physiology, and ecology of environmental microorganisms. Current challenges for metagenomic analysis are related to our ability to connect the dots between sequencing reads, their population of origin, and their encoding functions. Assembly-based methods reduce dataset size by extending overlapping reads into larger contiguous sequences (contigs, providing contextual information for genetic sequences that does not rely on existing references. These methods, however, tend to be computationally intensive and are again challenged by sequencing errors as well as by genomic repeats While numerous tools have been developed based on these methodological concepts, they present confounding choices and training requirements to metagenomic investigators. To help with accessibility to assembly tools, this review also includes an IPython Notebook metagenomic assembly tutorial. This tutorial has instructions for execution any operating system using Amazon Elastic Cloud Compute and guides users through downloading, assembly, and mapping reads to contigs of a mock microbiome metagenome. Despite its challenges, metagenomic analysis has already revealed novel insights into many environments on Earth. As software, training, and data continue to emerge, metagenomic data access and its discoveries will to grow.

A multi-substrate approach for functional metagenomics-based screening for (hemi)cellulases in two wheat straw-degrading microbial consortia unveils novel thermoalkaliphilic enzymes.

Science.gov (United States)

Maruthamuthu, Mukil; Jiménez, Diego Javier; Stevens, Patricia; van Elsas, Jan Dirk

2016-01-28

Functional metagenomics is a promising strategy for the exploration of the biocatalytic potential of microbiomes in order to uncover novel enzymes for industrial processes (e.g. biorefining or bleaching pulp). Most current methodologies used to screen for enzymes involved in plant biomass degradation are based on the use of single substrates. Moreover, highly diverse environments are used as metagenomic sources. However, such methods suffer from low hit rates of positive clones and hence the discovery of novel enzymatic activities from metagenomes has been hampered. Here, we constructed fosmid libraries from two wheat straw-degrading microbial consortia, denoted RWS (bred on untreated wheat straw) and TWS (bred on heat-treated wheat straw). Approximately 22,000 clones from each library were screened for (hemi)cellulose-degrading enzymes using a multi-chromogenic substrate approach. The screens yielded 71 positive clones for both libraries, giving hit rates of 1:440 and 1:1,047 for RWS and TWS, respectively. Seven clones (NT2-2, T5-5, NT18-17, T4-1, 10BT, NT18-21 and T17-2) were selected for sequence analyses. Their inserts revealed the presence of 18 genes encoding enzymes belonging to twelve different glycosyl hydrolase families (GH2, GH3, GH13, GH17, GH20, GH27, GH32, GH39, GH53, GH58, GH65 and GH109). These encompassed several carbohydrate-active gene clusters traceable mainly to Klebsiella related species. Detailed functional analyses showed that clone NT2-2 (containing a beta-galactosidase of ~116 kDa) had highest enzymatic activity at 55 °C and pH 9.0. Additionally, clone T5-5 (containing a beta-xylosidase of ~86 kDa) showed > 90% of enzymatic activity at 55 °C and pH 10.0. This study employed a high-throughput method for rapid screening of fosmid metagenomic libraries for (hemi)cellulose-degrading enzymes. The approach, consisting of screens on multi-substrates coupled to further analyses, revealed high hit rates, as compared with recent other studies. Two

Bioprospecting of Red Sea Sponges for Novel Antiviral Pharmacophores

KAUST Repository

O'Rourke, Aubrie

2015-05-01

Natural products offer many possibilities for the treatment of disease. More than 70% of the Earth’s surface is ocean, and recent exploration and access has allowed for new additions to this catalog of natural treasures. The Central Red Sea off the coast of Saudi Arabia serves as a newly accessible location, which provides the opportunity to bioprospect marine sponges with the purpose of identifying novel antiviral scaffolds. Antivirals are underrepresented in present day clinical trials, as well as in the academic screens of marine natural product libraries. Here a high-throughput pipeline was initiated by prefacing the antiviral screen with an Image-based High-Content Screening (HCS) technique in order to identify candidates with antiviral potential. Prospective candidates were tested in a biochemical or cell-based assay for the ability to inhibit the NS3 protease of the West Nile Virus (WNV NS protease) as well as replication and reverse transcription of the Human Immunodeficiency Virus 1 (HIV-1). The analytical chemistry techniques of High-Performance Liquid Chromatograpy (HPLC), Liquid Chromatography-Mass Spectrometry (LC-MS), and Nuclear Magnetic Resonance (NMR) where used in order to identify the compounds responsible for the characteristic antiviral activity of the selected sponge fractions. We have identified a 3-alkyl pyridinium from Amphimedon chloros as the causative agent of the observed WNV NS3 protease inhibition in vitro. Additionally, we identified debromohymenialdisine, hymenialdisine, and oroidin from Stylissa carteri as prospective scaffolds capable of HIV-1 inhibition.

HEK293T cell lines defective for O-linked glycosylation.

Directory of Open Access Journals (Sweden)

James M Termini

Full Text Available Here we describe derivatives of the HEK293T cell line that are defective in their ability to generate mucin-type O-linked glycosylation. Using CRISPR/Cas9 and a single-cell GFP-sorting procedure, the UDP-galactose-4-epimerase (GALE, galactokinase 1 (GALK1, and galactokinase 2 (GALK2 genes were knocked out individually and in combinations with greater than 90% of recovered clones having the desired mutations. Although HEK293T cells are tetraploid, we found this approach to be an efficient method to target and disrupt all 4 copies of the target gene. Deficient glycosylation in the GALE knockout cell line could be rescued by the addition of galactose and N-acetylgalactosamine (GalNAc to the cell culture media. However, when key enzymes of the galactose/GalNAc salvage pathways were disrupted in tandem (GALE+GALK1 or GALE+GALK2, O-glycosylation was eliminated and could not be rescued by the addition of either galactose plus GalNAc or UDP-galactose plus UDP-GalNAc. GALK1 and GALK2 are key enzymes of the galactose/GalNAc salvage pathways. Mass spectrometry was performed on whole cell lysate of the knockout cell lines to verify the glycosylation phenotype. As expected, the GALE knockout was almost completely devoid of all O-glycosylation, with minimal glycosylation as a result of functional salvage pathways. However, the GALE+GALK1 and GALE+GALK2 knockout lines were devoid of all O-glycans. Mass spectrometry analysis revealed that the disruption of GALE, GALK1, and GALE+GALK2 had little effect on the N-glycome. But when GALE was knocked out in tandem with GALK1, N-glycans were exclusively of the high mannose type. Due to the well-characterized nature of these five knockout cell lines, they will likely prove useful for a wide variety of applications.

Genomics and metagenomics in medical microbiology.

Science.gov (United States)

Padmanabhan, Roshan; Mishra, Ajay Kumar; Raoult, Didier; Fournier, Pierre-Edouard

2013-12-01

Over the last two decades, sequencing tools have evolved from laborious time-consuming methodologies to real-time detection and deciphering of genomic DNA. Genome sequencing, especially using next generation sequencing (NGS) has revolutionized the landscape of microbiology and infectious disease. This deluge of sequencing data has not only enabled advances in fundamental biology but also helped improve diagnosis, typing of pathogen, virulence and antibiotic resistance detection, and development of new vaccines and culture media. In addition, NGS also enabled efficient analysis of complex human micro-floras, both commensal, and pathological, through metagenomic methods, thus helping the comprehension and management of human diseases such as obesity. This review summarizes technological advances in genomics and metagenomics relevant to the field of medical microbiology. Copyright © 2013 Elsevier B.V. All rights reserved.

Glycosylation patterns of kidney proteins differ in rat diabetic nephropathy.

Science.gov (United States)

Ravidà, Alessandra; Musante, Luca; Kreivi, Marjut; Miinalainen, Ilkka; Byrne, Barry; Saraswat, Mayank; Henry, Michael; Meleady, Paula; Clynes, Martin; Holthofer, Harry

2015-05-01

Diabetic nephropathy often progresses to end-stage kidney disease and, ultimately, to renal replacement therapy. Hyperglycemia per se is expected to have a direct impact on the biosynthesis of N- and O-linked glycoproteins. This study aims to establish the link between protein glycosylation and progression of experimental diabetic kidney disease using orthogonal methods. Kidneys of streptozotocin-diabetic and control rats were harvested at three different time points post streptozotocin injection. A panel of 12 plant lectins was used in the screening of lectin blots. The lectins UEAI, PHA-E, GSI, PNA, and RCA identified remarkable disease-associated differences in glycoprotein expression. Lectin affinity chromatography followed by mass spectrometric analyses led to the identification of several glycoproteins involved in salt-handling, angiogenesis, and extracellular matrix degradation. Our data confirm a substantial link between glycosylation signature and diabetes progression. Furthermore, as suggested by our findings on dipeptidyl peptidase-IV, altered protein glycosylation may reflect changes in biochemical properties such as enzymatic activity. Thus, our study demonstrates the unexplored potential of protein glycosylation analysis in the discovery of molecules linked to diabetic kidney disease.

Model-based analysis of N-glycosylation in Chinese hamster ovary cells

DEFF Research Database (Denmark)

Krambeck, Frederick J.; Bennun, Sandra V; Andersen, Mikael Rørdam

2017-01-01

The Chinese hamster ovary (CHO) cell is the gold standard for manufacturing of glycosylated recombinant proteins for production of biotherapeutics. The similarity of its glycosylation patterns to the human versions enable the products of this cell line favorable pharmacokinetic properties and lower...

Oxytocin analogues with O-glycosylated serine and threonine in position 4

Czech Academy of Sciences Publication Activity Database

Marcinkowska, A.; Borovičková, Lenka; Slaninová, Jiřina; Grzonka, Z.

2007-01-01

Roč. 81, č. 7 (2007), s. 1335-1344 ISSN 0137- 5083 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z90210515 Keywords : oxytocin * glycosylated serin * glycosylated threonin * position 4 Subject RIV: CE - Biochemistry Impact factor: 0.483, year: 2007

Micropinocytic ingestion of glycosylated albumin by isolated microvessels: possible role in pathogenesis of diabetic microangiopathy.

OpenAIRE

Williams, S K; Devenny, J J; Bitensky, M W

1981-01-01

Microvessels isolated from rat epididymal fat exhibit differential vesicular ingestion rates for unmodified and non-enzymatically glycosylated rat albumin. While unmodified rat albumin is excluded from ingestion by endothelial micropinocytic vesicles, glycosylated albumin is avidly taken up by endocytosis. Interaction of albumin and glycosylated albumin with endothelium was studied with a double-label fluorescence assay of micropinocytosis. When glycosylated albumin was present at a concentra...

Comparative analysis of metagenomes of Italian top soil improvers

International Nuclear Information System (INIS)

Gigliucci, Federica; Brambilla, Gianfranco; Tozzoli, Rosangela; Michelacci, Valeria; Morabito, Stefano

2017-01-01

Biosolids originating from Municipal Waste Water Treatment Plants are proposed as top soil improvers (TSI) for their beneficial input of organic carbon on agriculture lands. Their use to amend soil is controversial, as it may lead to the presence of emerging hazards of anthropogenic or animal origin in the environment devoted to food production. In this study, we used a shotgun metagenomics sequencing as a tool to perform a characterization of the hazards related with the TSIs. The samples showed the presence of many virulence genes associated to different diarrheagenic E. coli pathotypes as well as of different antimicrobial resistance-associated genes. The genes conferring resistance to Fluoroquinolones was the most relevant class of antimicrobial resistance genes observed in all the samples tested. To a lesser extent traits associated with the resistance to Methicillin in Staphylococci and genes conferring resistance to Streptothricin, Fosfomycin and Vancomycin were also identified. The most represented metal resistance genes were cobalt-zinc-cadmium related, accounting for 15–50% of the sequence reads in the different metagenomes out of the total number of those mapping on the class of resistance to compounds determinants. Moreover the taxonomic analysis performed by comparing compost-based samples and biosolids derived from municipal sewage-sludges treatments divided the samples into separate populations, based on the microbiota composition. The results confirm that the metagenomics is efficient to detect genomic traits associated with pathogens and antimicrobial resistance in complex matrices and this approach can be efficiently used for the traceability of TSI samples using the microorganisms’ profiles as indicators of their origin. - Highlights: • Sludge- and green- based biosolids analysed by metagenomics. • Biosolids may introduce microbial hazards in the food chain. • Metagenomics enables tracking biosolids’ sources.

Comparative analysis of metagenomes of Italian top soil improvers

Energy Technology Data Exchange (ETDEWEB)

Gigliucci, Federica, E-mail: Federica.gigliucci@libero.it [Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Viale Regina Elena, 299 00161 Rome (Italy); Department of Sciences, University Roma,Tre, Viale Marconi, 446, 00146 Rome (Italy); Brambilla, Gianfranco; Tozzoli, Rosangela; Michelacci, Valeria; Morabito, Stefano [Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Viale Regina Elena, 299 00161 Rome (Italy)

2017-05-15

Biosolids originating from Municipal Waste Water Treatment Plants are proposed as top soil improvers (TSI) for their beneficial input of organic carbon on agriculture lands. Their use to amend soil is controversial, as it may lead to the presence of emerging hazards of anthropogenic or animal origin in the environment devoted to food production. In this study, we used a shotgun metagenomics sequencing as a tool to perform a characterization of the hazards related with the TSIs. The samples showed the presence of many virulence genes associated to different diarrheagenic E. coli pathotypes as well as of different antimicrobial resistance-associated genes. The genes conferring resistance to Fluoroquinolones was the most relevant class of antimicrobial resistance genes observed in all the samples tested. To a lesser extent traits associated with the resistance to Methicillin in Staphylococci and genes conferring resistance to Streptothricin, Fosfomycin and Vancomycin were also identified. The most represented metal resistance genes were cobalt-zinc-cadmium related, accounting for 15–50% of the sequence reads in the different metagenomes out of the total number of those mapping on the class of resistance to compounds determinants. Moreover the taxonomic analysis performed by comparing compost-based samples and biosolids derived from municipal sewage-sludges treatments divided the samples into separate populations, based on the microbiota composition. The results confirm that the metagenomics is efficient to detect genomic traits associated with pathogens and antimicrobial resistance in complex matrices and this approach can be efficiently used for the traceability of TSI samples using the microorganisms’ profiles as indicators of their origin. - Highlights: • Sludge- and green- based biosolids analysed by metagenomics. • Biosolids may introduce microbial hazards in the food chain. • Metagenomics enables tracking biosolids’ sources.

N-glycosylated catalytic unit meets O-glycosylated propeptide: complex protein architecture in a fungal hexosaminidase

Czech Academy of Sciences Publication Activity Database

Plíhal, Ondřej; Sklenář, Jan; Kmoníčková, J.; Man, Petr; Pompach, Petr; Havlíček, Vladimír; Křen, Vladimír; Bezouška, Karel

2004-01-01

Roč. 32, č. 5 (2004), s. 764-765 ISSN 0300-5127 R&D Projects: GA ČR GA203/04/1045 Institutional research plan: CEZ:AV0Z5020903 Keywords : asperillus oryzoe * glycosyl hydrolase * preproprotein Subject RIV: EE - Microbiology, Virology Impact factor: 2.267, year: 2004

A Delphi Technology Foresight Study: Mapping Social Construction of Scientific Evidence on Metagenomics Tests for Water Safety.

Directory of Open Access Journals (Sweden)

Stanislav Birko

Full Text Available Access to clean water is a grand challenge in the 21st century. Water safety testing for pathogens currently depends on surrogate measures such as fecal indicator bacteria (e.g., E. coli. Metagenomics concerns high-throughput, culture-independent, unbiased shotgun sequencing of DNA from environmental samples that might transform water safety by detecting waterborne pathogens directly instead of their surrogates. Yet emerging innovations such as metagenomics are often fiercely contested. Innovations are subject to shaping/construction not only by technology but also social systems/values in which they are embedded, such as experts' attitudes towards new scientific evidence. We conducted a classic three-round Delphi survey, comprised of 107 questions. A multidisciplinary expert panel (n = 24 representing the continuum of discovery scientists and policymakers evaluated the emergence of metagenomics tests. To the best of our knowledge, we report here the first Delphi foresight study of experts' attitudes on (1 the top 10 priority evidentiary criteria for adoption of metagenomics tests for water safety, (2 the specific issues critical to governance of metagenomics innovation trajectory where there is consensus or dissensus among experts, (3 the anticipated time lapse from discovery to practice of metagenomics tests, and (4 the role and timing of public engagement in development of metagenomics tests. The ability of a test to distinguish between harmful and benign waterborne organisms, analytical/clinical sensitivity, and reproducibility were the top three evidentiary criteria for adoption of metagenomics. Experts agree that metagenomic testing will provide novel information but there is dissensus on whether metagenomics will replace the current water safety testing methods or impact the public health end points (e.g., reduction in boil water advisories. Interestingly, experts view the publics relevant in a "downstream capacity" for adoption of

MOCAT: a metagenomics assembly and gene prediction toolkit.

Science.gov (United States)

Kultima, Jens Roat; Sunagawa, Shinichi; Li, Junhua; Chen, Weineng; Chen, Hua; Mende, Daniel R; Arumugam, Manimozhiyan; Pan, Qi; Liu, Binghang; Qin, Junjie; Wang, Jun; Bork, Peer

2012-01-01

MOCAT is a highly configurable, modular pipeline for fast, standardized processing of single or paired-end sequencing data generated by the Illumina platform. The pipeline uses state-of-the-art programs to quality control, map, and assemble reads from metagenomic samples sequenced at a depth of several billion base pairs, and predict protein-coding genes on assembled metagenomes. Mapping against reference databases allows for read extraction or removal, as well as abundance calculations. Relevant statistics for each processing step can be summarized into multi-sheet Excel documents and queryable SQL databases. MOCAT runs on UNIX machines and integrates seamlessly with the SGE and PBS queuing systems, commonly used to process large datasets. The open source code and modular architecture allow users to modify or exchange the programs that are utilized in the various processing steps. Individual processing steps and parameters were benchmarked and tested on artificial, real, and simulated metagenomes resulting in an improvement of selected quality metrics. MOCAT can be freely downloaded at http://www.bork.embl.de/mocat/.

MOCAT: a metagenomics assembly and gene prediction toolkit.

Directory of Open Access Journals (Sweden)

Jens Roat Kultima

Full Text Available MOCAT is a highly configurable, modular pipeline for fast, standardized processing of single or paired-end sequencing data generated by the Illumina platform. The pipeline uses state-of-the-art programs to quality control, map, and assemble reads from metagenomic samples sequenced at a depth of several billion base pairs, and predict protein-coding genes on assembled metagenomes. Mapping against reference databases allows for read extraction or removal, as well as abundance calculations. Relevant statistics for each processing step can be summarized into multi-sheet Excel documents and queryable SQL databases. MOCAT runs on UNIX machines and integrates seamlessly with the SGE and PBS queuing systems, commonly used to process large datasets. The open source code and modular architecture allow users to modify or exchange the programs that are utilized in the various processing steps. Individual processing steps and parameters were benchmarked and tested on artificial, real, and simulated metagenomes resulting in an improvement of selected quality metrics. MOCAT can be freely downloaded at http://www.bork.embl.de/mocat/.

Automated and Accurate Estimation of Gene Family Abundance from Shotgun Metagenomes.

Directory of Open Access Journals (Sweden)

Stephen Nayfach

2015-11-01

Full Text Available 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.

Ruminal metagenomic libraries as a source of relevant hemicellulolytic enzymes for biofuel production.

Science.gov (United States)

Duque, Estrella; Daddaoua, Abdelali; Cordero, Baldo F; Udaondo, Zulema; Molina-Santiago, Carlos; Roca, Amalia; Solano, Jennifer; Molina-Alcaide, Eduarda; Segura, Ana; Ramos, Juan-Luis

2018-04-17

The success of second-generation (2G) ethanol technology relies on the efficient transformation of hemicellulose into monosaccharides and, particularly, on the full conversion of xylans into xylose for over 18% of fermentable sugars. We sought new hemicellulases using ruminal liquid, after enrichment of microbes with industrial lignocellulosic substrates and preparation of metagenomic libraries. Among 150 000 fosmid clones tested, we identified 22 clones with endoxylanase activity and 125 with β-xylosidase activity. These positive clones were sequenced en masse, and the analysis revealed open reading frames with a low degree of similarity with known glycosyl hydrolases families. Among them, we searched for enzymes that were thermostable (activity at > 50°C) and that operate at high rate at pH around 5. Upon a wide series of assays, the clones exhibiting the highest endoxylanase and β-xylosidase activities were identified. The fosmids were sequenced, and the corresponding genes cloned, expressed and proteins purified. We found that the activity of the most active β-xylosidase was at least 10-fold higher than that in commercial enzymatic fungal cocktails. Endoxylanase activity was in the range of fungal enzymes. Fungal enzymatic cocktails supplemented with the bacterial hemicellulases exhibited enhanced release of sugars from pretreated sugar cane straw, a relevant agricultural residue. © 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

In-depth mapping of the mouse brain N-glycoproteome reveals widespread N-glycosylation of diverse brain proteins.

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Fang, Pan; Wang, Xin-Jian; Xue, Yu; Liu, Ming-Qi; Zeng, Wen-Feng; Zhang, Yang; Zhang, Lei; Gao, Xing; Yan, Guo-Quan; Yao, Jun; Shen, Hua-Li; Yang, Peng-Yuan

2016-06-21

N-glycosylation is one of the most prominent and abundant posttranslational modifications of proteins. It is estimated that over 50% of mammalian proteins undergo glycosylation. However, the analysis of N-glycoproteins has been limited by the available analytical technology. In this study, we comprehensively mapped the N-glycosylation sites in the mouse brain proteome by combining complementary methods, which included seven protease treatments, four enrichment techniques and two fractionation strategies. Altogether, 13492 N-glycopeptides containing 8386 N-glycosylation sites on 3982 proteins were identified. After evaluating the performance of the above methods, we proposed a simple and efficient workflow for large-scale N-glycosylation site mapping. The optimized workflow yielded 80% of the initially identified N-glycosylation sites with considerably less effort. Analysis of the identified N-glycoproteins revealed that many of the mouse brain proteins are N-glycosylated, including those proteins in critical pathways for nervous system development and neurological disease. Additionally, several important biomarkers of various diseases were found to be N-glycosylated. These data confirm that N-glycosylation is important in both physiological and pathological processes in the brain, and provide useful details about numerous N-glycosylation sites in brain proteins.

A retrospective metagenomics approach to studying Blastocystis.

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Andersen, Lee O'Brien; Bonde, Ida; Nielsen, Henrik Bjørn; Stensvold, Christen Rune

2015-07-01

Blastocystis is a common single-celled intestinal parasitic genus, comprising several subtypes. Here, we screened data obtained by metagenomic analysis of faecal DNA for Blastocystis by searching for subtype-specific genes in coabundance gene groups, which are groups of genes that covary across a selection of 316 human faecal samples, hence representing genes originating from a single subtype. The 316 faecal samples were from 236 healthy individuals, 13 patients with Crohn's disease (CD) and 67 patients with ulcerative colitis (UC). The prevalence of Blastocystis was 20.3% in the healthy individuals and 14.9% in patients with UC. Meanwhile, Blastocystis was absent in patients with CD. Individuals with intestinal microbiota dominated by Bacteroides were much less prone to having Blastocystis-positive stool (Matthew's correlation coefficient = -0.25, P < 0.0001) than individuals with Ruminococcus- and Prevotella-driven enterotypes. This is the first study to investigate the relationship between Blastocystis and communities of gut bacteria using a metagenomics approach. The study serves as an example of how it is possible to retrospectively investigate microbial eukaryotic communities in the gut using metagenomic datasets targeting the bacterial component of the intestinal microbiome and the interplay between these microbial communities. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Fab glycosylation of immunoglobulin G does not associate with improvement of rheumatoid arthritis during pregnancy.

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Bondt, Albert; Wuhrer, Manfred; Kuijper, T Martijn; Hazes, Johanna M W; Dolhain, Radboud J E M

2016-11-25

Changes in immunoglobulin G (IgG) constant domain (Fc) glycosylation are associated with changes in rheumatoid arthritis (RA) disease activity in response to pregnancy. Here, we sought to determine whether the same holds true for variable domain (Fab) glycosylation. IgGs were captured from RA and control sera obtained before (RA only), during and after pregnancy, followed by Fc and Fab separation, glycan release, and mass spectrometric detection. In parallel, glycans from intact IgG were analysed. The data was used to calculate glycosylation traits, and to estimate the level of Fab glycosylation. The overall level of Fab glycosylation was increased in RA patients compared to controls, while no differences in Fab glycosylation patterns were found. For the Fc and intact IgG (Total) previously observed differences in galactosylation and bisection were confirmed. Furthermore, increased galactosylation of Fc and Total were associated with lower disease activity and autoantibody positivity. In addition, the change in Fc galactosylation associated with the change in disease activity during pregnancy and after delivery, while this was not the case for Fab. In contrast to changes in Fc glycosylation, changes in Fab glycosylation are not associated with improvement of RA during pregnancy and arthritis flare after delivery.

Metagenomic-based study of the phylogenetic and functional gene diversity in Galápagos land and marine iguanas.

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Hong, Pei-Ying; Mao, Yuejian; Ortiz-Kofoed, Shannon; Shah, Rushabh; Cann, Isaac; Mackie, Roderick I

2015-02-01

In this study, a metagenome-based analysis of the fecal samples from the macrophytic algae-consuming marine iguana (MI; Amblyrhynchus cristatus) and terrestrial biomass-consuming land iguanas (LI; Conolophus spp.) was conducted. Phylogenetic affiliations of the fecal microbiome were more similar between both iguanas than to other mammalian herbivorous hosts. However, functional gene diversities in both MI and LI iguana hosts differed in relation to the diet, where the MI fecal microbiota had a functional diversity that clustered apart from the other terrestrial-biomass consuming reptilian and mammalian hosts. A further examination of the carbohydrate-degrading genes revealed that several of the prevalent glycosyl hydrolases (GH), glycosyl transferases (GT), carbohydrate binding modules (CBM), and carbohydrate esterases (CE) gene classes were conserved among all examined herbivorous hosts, reiterating the important roles these genes play in the breakdown and metabolism of herbivorous diets. Genes encoding some classes of carbohydrate-degrading families, including GH2, GH13, GT2, GT4, CBM50, CBM48, CE4, and CE11, as well as genes associated with sulfur metabolism and dehalogenation, were highly enriched or unique to the MI. In contrast, gene sequences that relate to archaeal methanogenesis were detected only in LI fecal microbiome, and genes coding for GH13, GH66, GT2, GT4, CBM50, CBM13, CE4, and CE8 carbohydrate active enzymes were highly abundant in the LI. Bacterial populations were enriched on various carbohydrates substrates (e.g., glucose, arabinose, xylose). The majority of the enriched bacterial populations belong to genera Clostridium spp. and Enterococcus spp. that likely accounted for the high prevalence of GH13 and GH2, as well as the GT families (e.g., GT2, GT4, GT28, GT35, and GT51) that were ubiquitously present in the fecal microbiota of all herbivorous hosts.

Metagenomic-Based Study of the Phylogenetic and Functional Gene Diversity in Galápagos Land and Marine Iguanas

KAUST Repository

Hong, Pei-Ying

2014-12-19

In this study, a metagenome-based analysis of the fecal samples from the macrophytic algae-consuming marine iguana (MI; Amblyrhynchus cristatus) and terrestrial biomass-consuming land iguanas (LI; Conolophus spp.) was conducted. Phylogenetic affiliations of the fecal microbiome were more similar between both iguanas than to other mammalian herbivorous hosts. However, functional gene diversities in both MI and LI iguana hosts differed in relation to the diet, where the MI fecal microbiota had a functional diversity that clustered apart from the other terrestrial-biomass consuming reptilian and mammalian hosts. A further examination of the carbohydrate-degrading genes revealed that several of the prevalent glycosyl hydrolases (GH), glycosyl transferases (GT), carbohydrate binding modules (CBM), and carbohydrate esterases (CE) gene classes were conserved among all examined herbivorous hosts, reiterating the important roles these genes play in the breakdown and metabolism of herbivorous diets. Genes encoding some classes of carbohydrate-degrading families, including GH2, GH13, GT2, GT4, CBM50, CBM48, CE4, and CE11, as well as genes associated with sulfur metabolism and dehalogenation, were highly enriched or unique to the MI. In contrast, gene sequences that relate to archaeal methanogenesis were detected only in LI fecal microbiome, and genes coding for GH13, GH66, GT2, GT4, CBM50, CBM13, CE4, and CE8 carbohydrate active enzymes were highly abundant in the LI. Bacterial populations were enriched on various carbohydrates substrates (e.g., glucose, arabinose, xylose). The majority of the enriched bacterial populations belong to genera Clostridium spp. and Enterococcus spp. that likely accounted for the high prevalence of GH13 and GH2, as well as the GT families (e.g., GT2, GT4, GT28, GT35, and GT51) that were ubiquitously present in the fecal microbiota of all herbivorous hosts.

Analysis of expression and glycosylation of avian metapneumovirus attachment glycoprotein from recombinant baculoviruses.

Science.gov (United States)

Luo, Lizhong; Nishi, Krista; MacLeod, Erin; Sabara, Marta I; Li, Yan

2010-11-01

Recently, we reported the expression and glycosylation of avian metapneumovirus attachment glycoprotein (AMPV/C G protein) in eukaryotic cell lines by a transient-expression method. In the present study, we investigated the biosynthesis and O-linked glycosylation of the AMPV/C G protein in a baculovirus expression system. The results showed that the insect cell-produced G protein migrated more rapidly in SDS-PAGE as compared to LLC-MK2 cell-derived G proteins owing to glycosylation differences. The fully processed, mature form of G protein migrated between 78 and 86 kDa, which is smaller than the 110 kDa mature form of G expressed in LLC-MK2 cells. In addition, several immature G gene products migrating at 40-48 and 60-70 kDa were also detected by SDS-PAGE and represented glycosylated intermediates. The addition of the antibiotic tunicamycin, which blocks early steps of glycosylation, to insect cell culture resulted in the disappearance of two glycosylated forms of the G protein and identified a 38 kDa unglycosylated precursor. The maturation of the G protein was completely blocked by monensin, suggesting that the O-linked glycosylation of G initiated in the trans-Golgi compartment. The presence of O-linked sugars on the mature protein was further confirmed by lectin Arachis hypogaea binding assay. Furthermore, antigenic features of the G protein expressed in insect cells were evaluated by ELISA. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

Resolving the Complexity of Human Skin Metagenomes Using Single-Molecule Sequencing

Directory of Open Access Journals (Sweden)

Yu-Chih Tsai

2016-02-01

Full Text Available Deep metagenomic shotgun sequencing has emerged as a powerful tool to interrogate composition and function of complex microbial communities. Computational approaches to assemble genome fragments have been demonstrated to be an effective tool for de novo reconstruction of genomes from these communities. However, the resultant “genomes” are typically fragmented and incomplete due to the limited ability of short-read sequence data to assemble complex or low-coverage regions. Here, we use single-molecule, real-time (SMRT sequencing to reconstruct a high-quality, closed genome of a previously uncharacterized Corynebacterium simulans and its companion bacteriophage from a skin metagenomic sample. Considerable improvement in assembly quality occurs in hybrid approaches incorporating short-read data, with even relatively small amounts of long-read data being sufficient to improve metagenome reconstruction. Using short-read data to evaluate strain variation of this C. simulans in its skin community at single-nucleotide resolution, we observed a dominant C. simulans strain with moderate allelic heterozygosity throughout the population. We demonstrate the utility of SMRT sequencing and hybrid approaches in metagenome quantitation, reconstruction, and annotation.

Resolving the Complexity of Human Skin Metagenomes Using Single-Molecule Sequencing

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Tsai, Yu-Chih; Deming, Clayton; Segre, Julia A.; Kong, Heidi H.; Korlach, Jonas

2016-01-01

ABSTRACT Deep metagenomic shotgun sequencing has emerged as a powerful tool to interrogate composition and function of complex microbial communities. Computational approaches to assemble genome fragments have been demonstrated to be an effective tool for de novo reconstruction of genomes from these communities. However, the resultant “genomes” are typically fragmented and incomplete due to the limited ability of short-read sequence data to assemble complex or low-coverage regions. Here, we use single-molecule, real-time (SMRT) sequencing to reconstruct a high-quality, closed genome of a previously uncharacterized Corynebacterium simulans and its companion bacteriophage from a skin metagenomic sample. Considerable improvement in assembly quality occurs in hybrid approaches incorporating short-read data, with even relatively small amounts of long-read data being sufficient to improve metagenome reconstruction. Using short-read data to evaluate strain variation of this C. simulans in its skin community at single-nucleotide resolution, we observed a dominant C. simulans strain with moderate allelic heterozygosity throughout the population. We demonstrate the utility of SMRT sequencing and hybrid approaches in metagenome quantitation, reconstruction, and annotation. PMID:26861018

Cyclodipeptides from metagenomic library of a japanese marine sponge

Energy Technology Data Exchange (ETDEWEB)

He, Rui; Wang, Bochu; Zhub, Liancai, E-mail: wangbc2000@126.com [Bioengineering College, Chongqing University, Chongqing, (China); Wang, Manyuan [School of Traditional Chinese Medicine, Capital University of Medical Sciences, Beijing (China); Wakimoto, Toshiyuki; Abe, Ikuro, E-mail: abei@mol.f.u-tokyo.ac.jp [Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo (Japan)

2013-12-01

Culture-independent metagenomics is an attractive and promising approach to explore unique bioactive small molecules from marine sponges harboring uncultured symbiotic microbes. Therefore, we conducted functional screening of the metagenomic library constructed from the Japanese marine sponge Discodermia calyx. Bioassay-guided fractionation of plate culture extract of antibacterial clone pDC113 afforded eleven cyclodipeptides: Cyclo(l-Thr-l-Leu) (1), Cyclo(l-Val-d-Pro) (2), Cyclo(l-Ile-d-Pro) (3), Cyclo(l-Leu-l-Pro) (4), Cyclo(l-Val-l-Leu) (5), Cyclo(l-Leu-l-Ile) (6), Cyclo(l-Leu-l-Leu) (7), Cyclo(l-Phe-l-Tyr) (8), Cyclo(l-Trp-l-Pro) (9), Cyclo(l-Val-l-Trp) (10) and Cyclo(l-Ile-l-Trp) (11). To the best of our knowledge, these are first cyclodepeptides isolated from metagenomic library. Sequence analysis suggested that isolated cyclodipeptides were not synthesized by nonribosomal peptide synthetases and there was no significant indication of cyclodipeptide synthetases. (author)

Cyclodipeptides from metagenomic library of a japanese marine sponge

International Nuclear Information System (INIS)

He, Rui; Wang, Bochu; Zhub, Liancai; Wang, Manyuan; Wakimoto, Toshiyuki; Abe, Ikuro

2013-01-01

Culture-independent metagenomics is an attractive and promising approach to explore unique bioactive small molecules from marine sponges harboring uncultured symbiotic microbes. Therefore, we conducted functional screening of the metagenomic library constructed from the Japanese marine sponge Discodermia calyx. Bioassay-guided fractionation of plate culture extract of antibacterial clone pDC113 afforded eleven cyclodipeptides: Cyclo(l-Thr-l-Leu) (1), Cyclo(l-Val-d-Pro) (2), Cyclo(l-Ile-d-Pro) (3), Cyclo(l-Leu-l-Pro) (4), Cyclo(l-Val-l-Leu) (5), Cyclo(l-Leu-l-Ile) (6), Cyclo(l-Leu-l-Leu) (7), Cyclo(l-Phe-l-Tyr) (8), Cyclo(l-Trp-l-Pro) (9), Cyclo(l-Val-l-Trp) (10) and Cyclo(l-Ile-l-Trp) (11). To the best of our knowledge, these are first cyclodepeptides isolated from metagenomic library. Sequence analysis suggested that isolated cyclodipeptides were not synthesized by nonribosomal peptide synthetases and there was no significant indication of cyclodipeptide synthetases. (author)

N-Glycosylation optimization of recombinant antibodies in CHO cell through process and metabolic engineering

DEFF Research Database (Denmark)

Fan, Yuzhou

, analysis, control and optimization of N-glycosylation were thoroughly reviewed. In particular, how to control and optimize N-glycosylation in CHO cells was exclusively studied. The main focus of this PhD project is to find effective approaches of modulating N-glycosylation of CHO-derived recombinant...... galactose as feed additives, changing process parameters such as seeding density and cultivation duration are all demonstrated to be effective. The causal explanation of their impact on glycosylation can be various, including product, metabolism, proteome and physiology-associated mechanism. In the middle...... part of the thesis, both literature reviews and experimental applications were provided to demonstrate how to use omics data and implement systems biology to understand biological activities, especially N-glycosylation in CHO cells. In the last part of the thesis, the second strategy that apply genetic...

Enzymatic glycosylation of multivalent scaffolds

Czech Academy of Sciences Publication Activity Database

Bojarová, Pavla; Rosencrantz, R. R.; Elling, L.; Křen, Vladimír

2013-01-01

Roč. 42, č. 11 (2013), s. 4774-4797 ISSN 0306-0012 R&D Projects: GA MŠk(CZ) LD13042; GA ČR GAP207/10/0321 Institutional support: RVO:61388971 Keywords : N-ACETYLGLUCOSAMINYLTRANSFERASE-III * MUCIN TANDEM REPEAT * NEIGHBORING RESIDUE GLYCOSYLATION Subject RIV: CC - Organic Chemistry Impact factor: 30.425, year: 2013

COMPARISON OF FRUCTOSAMINE AND GLYCOSYLATED HEMOGLOBIN IN A NON-INSULIN DEPENDENT DIABETIC POPULATION

Directory of Open Access Journals (Sweden)

M. Amini

1999-08-01

Full Text Available In an attempt to determine the clinical value of frnctosamine assay for monitoring type II diabetic patients, correlation of frnctosamine with glycosylated hemoglobin was studied. 100 patients with type II diabetes mcllitus were compared with 100 normal subjects. Fasting blood glucose, glycosylated hemoglobin, albumin and frnctosamine were measured in alt subjects. In the diabetic patients, a significant correlation was observed between fasting blood glucose and glycosylated hemoglobin (r = 0.64, p < 0.01 and scrum frnctosamine (r = 0.7, P < 0.01. Tlicrc was also a significant correlation between glycosylated hemoglobin and scrum frtictosmine (r = .94, I'<0.01. Frnctosamine, assay can be used as an index of diabetes control.

Bioprospection of immature salivary glands of Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae).

Science.gov (United States)

Caleffe, Ronaldo Roberto Tait; de Oliveira, Stefany Rodrigues; Gigliolli, Adriana Aparecida Sinópolis; Ruvolo-Takasusuki, Maria Claudia Colla; Conte, Helio

2018-06-08

Larval therapy (LT) comprises the application of sterile Calliphoridae larvae for wound debridement, disinfection, and healing in humans and animals. Larval digestion plays a key role in LT, where the salivary glands and gut produce and secrete proteolytic and antimicrobial substances. The objective of this work was to bioprospect the salivary glands of Chrysomya megacephala (Fabricius, 1794) larvae, using ultrastructural, morphological, and histological observations, and the total protein electrophoretic profile. The salivary glands present a deferent duct, originating from the buccal cavity, which bifurcates into efferent ducts that insert through a slight dilatation to a pair of tubular-shaped tissues, united in the region of fat cells. Histologically, the secretion had protein characteristics. Cell cytoplasm presented numerous free ribosomes, autophagic vacuoles, spherical and elongated mitochondria, atypical Golgi complexes, and dilated rough endoplasmic reticulum. In the apical cytoplasm, secretory granules and microvilli secretions demonstrated intense protein synthesis, basal cytoplasm with trachea insertions, and numerous mitochondria. The present work described the ultrastructure and morphology of C. megacephala third instar salivary glands, confirming intense protein synthesis and the molecular weight of soluble proteins. Copyright © 2018 Elsevier Ltd. All rights reserved.

ELIXIR pilot action: Marine metagenomics – towards a domain specific set of sustainable services

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Robertsen, Espen Mikal; Denise, Hubert; Mitchell, Alex; Finn, Robert D.; Bongo, Lars Ailo; Willassen, Nils Peder

2017-01-01

Metagenomics, the study of genetic material recovered directly from environmental samples, has the potential to provide insight into the structure and function of heterogeneous microbial communities.  There has been an increased use of metagenomics to discover and understand the diverse biosynthetic capacities of marine microbes, thereby allowing them to be exploited for industrial, food, and health care products. This ELIXIR pilot action was motivated by the need to establish dedicated data resources and harmonized metagenomics pipelines for the marine domain, in order to enhance the exploration and exploitation of marine genetic resources. In this paper, we summarize some of the results from the ELIXIR pilot action “Marine metagenomics – towards user centric services”. PMID:28620454

Importance of glycosylation on function of a potassium channel in neuroblastoma cells.

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M K Hall

Full Text Available The Kv3.1 glycoprotein, a voltage-gated potassium channel, is expressed throughout the central nervous system. The role of N-glycans attached to the Kv3.1 glycoprotein on conducting and non-conducting functions of the Kv3.1 channel are quite limiting. Glycosylated (wild type, partially glycosylated (N220Q and N229Q, and unglycosylated (N220Q/N229Q Kv3.1 proteins were expressed and characterized in a cultured neuronal-derived cell model, B35 neuroblastoma cells. Western blots, whole cell current recordings, and wound healing assays were employed to provide evidence that the conducting and non-conducting properties of the Kv3.1 channel were modified by N-glycans of the Kv3.1 glycoprotein. Electrophoretic migration of the various Kv3.1 proteins treated with PNGase F and neuraminidase verified that the glycosylation sites were occupied and that the N-glycans could be sialylated, respectively. The unglycosylated channel favored a different whole cell current pattern than the glycoform. Further the outward ionic currents of the unglycosylated channel had slower activation and deactivation rates than those of the glycosylated Kv3.1 channel. These kinetic parameters of the partially glycosylated Kv3.1 channels were also slowed. B35 cells expressing glycosylated Kv3.1 protein migrated faster than those expressing partially glycosylated and much faster than those expressing the unglycosylated Kv3.1 protein. These results have demonstrated that N-glycans of the Kv3.1 glycoprotein enhance outward ionic current kinetics, and neuronal migration. It is speculated that physiological changes which lead to a reduction in N-glycan attachment to proteins will alter the functions of the Kv3.1 channel.

Biological role of site-specific O-glycosylation in cell adhesion activity and phosphorylation of osteopontin.

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Oyama, Midori; Kariya, Yoshinobu; Kariya, Yukiko; Matsumoto, Kana; Kanno, Mayumi; Yamaguchi, Yoshiki; Hashimoto, Yasuhiro

2018-05-09

Osteopontin (OPN) is an extracellular glycosylated phosphoprotein that promotes cell adhesion by interacting with several integrin receptors. We previously reported that an OPN mutant lacking five O-glycosylation sites (Thr 134 /Thr 138 /Thr 143 /Thr 147 /Thr 152 ) in the threonine/proline-rich region increased cell adhesion activity and phosphorylation compared with the wild type. However, the role of O-glycosylation in cell adhesion activity and phosphorylation of OPN remains to be clarified. Here, we show that site-specific O-glycosylation in the threonine/proline-rich region of OPN affects its cell adhesion activity and phosphorylation independently and/or synergistically. Using site-directed mutagenesis, we found that OPN mutants with substitution sets of Thr 134 /Thr 138 or Thr 143 /Thr 147 /Thr 152 had decreased and increased cell adhesion activity, respectively. In contrast, the introduction of a single mutation into the O-glycosylation sites had no effect on OPN cell adhesion activity. An adhesion assay using function-blocking antibodies against αvβ3 and β1 integrins, as well as αvβ3 integrin-overexpressing A549 cells, revealed that site-specific O-glycosylation affected the association of OPN with the two integrins. Phosphorylation analyses using phos-tag and LC-MS/MS indicated that phosphorylation levels and sites were influenced by the O-glycosylation status, although the number of O-glycosylation sites was not correlated with the phosphorylation level in OPN. Furthermore, a correlation analysis between phosphorylation level and cell adhesion activity in OPN mutants with the site-specific O-glycosylation showed that they were not always correlated. These results provide conclusive evidence of a novel regulatory mechanism of cell adhesion activity and phosphorylation of OPN by site-specific O-glycosylation. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

MetaBAT: Metagenome Binning based on Abundance and Tetranucleotide frequence

Energy Technology Data Exchange (ETDEWEB)

Kang, Dongwan; Froula, Jeff; Egan, Rob; Wang, Zhong

2014-03-21

Grouping large fragments assembled from shotgun metagenomic sequences to deconvolute complex microbial communities, or metagenome binning, enables the study of individual organisms and their interactions. Here we developed automated metagenome binning software, called MetaBAT, which integrates empirical probabilistic distances of genome abundance and tetranucleotide frequency. On synthetic datasets MetaBAT on average achieves 98percent precision and 90percent recall at the strain level with 281 near complete unique genomes. Applying MetaBAT to a human gut microbiome data set we recovered 176 genome bins with 92percent precision and 80percent recall. Further analyses suggest MetaBAT is able to recover genome fragments missed in reference genomes up to 19percent, while 53 genome bins are novel. In summary, we believe MetaBAT is a powerful tool to facilitate comprehensive understanding of complex microbial communities.

MALINA: a web service for visual analytics of human gut microbiota whole-genome metagenomic reads.

Science.gov (United States)

Tyakht, Alexander V; Popenko, Anna S; Belenikin, Maxim S; Altukhov, Ilya A; Pavlenko, Alexander V; Kostryukova, Elena S; Selezneva, Oksana V; Larin, Andrei K; Karpova, Irina Y; Alexeev, Dmitry G

2012-12-07

MALINA is a web service for bioinformatic analysis of whole-genome metagenomic data obtained from human gut microbiota sequencing. As input data, it accepts metagenomic reads of various sequencing technologies, including long reads (such as Sanger and 454 sequencing) and next-generation (including SOLiD and Illumina). It is the first metagenomic web service that is capable of processing SOLiD color-space reads, to authors' knowledge. The web service allows phylogenetic and functional profiling of metagenomic samples using coverage depth resulting from the alignment of the reads to the catalogue of reference sequences which are built into the pipeline and contain prevalent microbial genomes and genes of human gut microbiota. The obtained metagenomic composition vectors are processed by the statistical analysis and visualization module containing methods for clustering, dimension reduction and group comparison. Additionally, the MALINA database includes vectors of bacterial and functional composition for human gut microbiota samples from a large number of existing studies allowing their comparative analysis together with user samples, namely datasets from Russian Metagenome project, MetaHIT and Human Microbiome Project (downloaded from http://hmpdacc.org). MALINA is made freely available on the web at http://malina.metagenome.ru. The website is implemented in JavaScript (using Ext JS), Microsoft .NET Framework, MS SQL, Python, with all major browsers supported.

Construction and screening of marine metagenomic libraries.

Science.gov (United States)

Weiland, Nancy; Löscher, Carolin; Metzger, Rebekka; Schmitz, Ruth

2010-01-01

Marine microbial communities are highly diverse and have evolved during extended evolutionary processes of physiological adaptations under the influence of a variety of ecological conditions and selection pressures. They harbor an enormous diversity of microbes with still unknown and probably new physiological characteristics. Besides, the surfaces of marine multicellular organisms are typically covered by a consortium of epibiotic bacteria and act as barriers, where diverse interactions between microorganisms and hosts take place. Thus, microbial diversity in the water column of the oceans and the microbial consortia on marine tissues of multicellular organisms are rich sources for isolating novel bioactive compounds and genes. Here we describe the sampling, construction of large-insert metagenomic libraries from marine habitats and exemplarily one function based screen of metagenomic clones.

Analysis of composition-based metagenomic classification.

Science.gov (United States)

Higashi, Susan; Barreto, André da Motta Salles; Cantão, Maurício Egidio; de Vasconcelos, Ana Tereza Ribeiro

2012-01-01

An essential step of a metagenomic study is the taxonomic classification, that is, the identification of the taxonomic lineage of the organisms in a given sample. The taxonomic classification process involves a series of decisions. Currently, in the context of metagenomics, such decisions are usually based on empirical studies that consider one specific type of classifier. In this study we propose a general framework for analyzing the impact that several decisions can have on the classification problem. Instead of focusing on any specific classifier, we define a generic score function that provides a measure of the difficulty of the classification task. Using this framework, we analyze the impact of the following parameters on the taxonomic classification problem: (i) the length of n-mers used to encode the metagenomic sequences, (ii) the similarity measure used to compare sequences, and (iii) the type of taxonomic classification, which can be conventional or hierarchical, depending on whether the classification process occurs in a single shot or in several steps according to the taxonomic tree. We defined a score function that measures the degree of separability of the taxonomic classes under a given configuration induced by the parameters above. We conducted an extensive computational experiment and found out that reasonable values for the parameters of interest could be (i) intermediate values of n, the length of the n-mers; (ii) any similarity measure, because all of them resulted in similar scores; and (iii) the hierarchical strategy, which performed better in all of the cases. As expected, short n-mers generate lower configuration scores because they give rise to frequency vectors that represent distinct sequences in a similar way. On the other hand, large values for n result in sparse frequency vectors that represent differently metagenomic fragments that are in fact similar, also leading to low configuration scores. Regarding the similarity measure, in

Tapping uncultured microorganisms through metagenomics for drug ...

African Journals Online (AJOL)

African Journal of Biotechnology ... Microorganisms are major source of bioactive natural products, and several ... This review highlights the recent methodologies, limitations, and applications of metagenomics for the discovery of new drugs.

Natural history bycatch: a pipeline for identifying metagenomic sequences in RADseq data

Directory of Open Access Journals (Sweden)

Iris Holmes

2018-04-01

Full Text Available Background Reduced representation genomic datasets are increasingly becoming available from a variety of organisms. These datasets do not target specific genes, and so may contain sequences from parasites and other organisms present in the target tissue sample. In this paper, we demonstrate that (1 RADseq datasets can be used for exploratory analysis of tissue-specific metagenomes, and (2 tissue collections house complete metagenomic communities, which can be investigated and quantified by a variety of techniques. Methods We present an exploratory method for mining metagenomic “bycatch” sequences from a range of host tissue types. We use a combination of the pyRAD assembly pipeline, NCBI’s blastn software, and custom R scripts to isolate metagenomic sequences from RADseq type datasets. Results When we focus on sequences that align with existing references in NCBI’s GenBank, we find that between three and five percent of identifiable double-digest restriction site associated DNA (ddRAD sequences from host tissue samples are from phyla to contain known blood parasites. In addition to tissue samples, we examine ddRAD sequences from metagenomic DNA extracted snake and lizard hind-gut samples. We find that the sequences recovered from these samples match with expected bacterial and eukaryotic gut microbiome phyla. Discussion Our results suggest that (1 museum tissue banks originally collected for host DNA archiving are also preserving valuable parasite and microbiome communities, (2 that publicly available RADseq datasets may include metagenomic sequences that could be explored, and (3 that restriction site approaches are a useful exploratory technique to identify microbiome lineages that could be missed by primer-based approaches.

Untangling Genomes from Metagenomes: Revealing an Uncultured Class of Marine Euryarchaeota

Science.gov (United States)

Iverson, Vaughn; Morris, Robert M.; Frazar, Christian D.; Berthiaume, Chris T.; Morales, Rhonda L.; Armbrust, E. Virginia

2012-02-01

Ecosystems are shaped by complex communities of mostly unculturable microbes. Metagenomes provide a fragmented view of such communities, but the ecosystem functions of major groups of organisms remain mysterious. To better characterize members of these communities, we developed methods to reconstruct genomes directly from mate-paired short-read metagenomes. We closed a genome representing the as-yet uncultured marine group II Euryarchaeota, assembled de novo from 1.7% of a metagenome sequenced from surface seawater. The genome describes a motile, photo-heterotrophic cell focused on degradation of protein and lipids and clarifies the origin of proteorhodopsin. It also demonstrates that high-coverage mate-paired sequence can overcome assembly difficulties caused by interstrain variation in complex microbial communities, enabling inference of ecosystem functions for uncultured members.

Assembly of viral genomes from metagenomes

NARCIS (Netherlands)

S.L. Smits (Saskia); R. Bodewes (Rogier); A. Ruiz-Gonzalez (Aritz); V. Baumgärtner (Volkmar); M.P.G. Koopmans D.V.M. (Marion); A.D.M.E. Osterhaus (Albert); A. Schürch (Anita)

2014-01-01

textabstractViral infections remain a serious global health issue. Metagenomic approaches are increasingly used in the detection of novel viral pathogens but also to generate complete genomes of uncultivated viruses. In silico identification of complete viral genomes from sequence data would allow

Metagenomes obtained by "deep sequencing" - what do they tell about the EBPR communities?

DEFF Research Database (Denmark)

Albertsen, Mads; Saunders, Aaron Marc; Nielsen, Kåre Lehmann

2013-01-01

Metagenomics enables studies of the genomic potential of complex microbial communities by sequencing bulk genomic DNA directly from the environment. Knowledge of the genetic potential of a community can be used to formulate and test ecological hypotheses about stability and performance...... demonstrate that metagenomics can be used as a powerful tool for system wide characterization of the EBPR community as well as for a deeper understanding of the function of specific community members. Furthermore, we discuss and illustrate some of the general pitfalls in metagenomics and stress the need...

Macrocyclic bis-thioureas catalyze stereospecific glycosylation reactions.

Science.gov (United States)

Park, Yongho; Harper, Kaid C; Kuhl, Nadine; Kwan, Eugene E; Liu, Richard Y; Jacobsen, Eric N

2017-01-13

Carbohydrates are involved in nearly all aspects of biochemistry, but their complex chemical structures present long-standing practical challenges to their synthesis. In particular, stereochemical outcomes in glycosylation reactions are highly dependent on the steric and electronic properties of coupling partners; thus, carbohydrate synthesis is not easily predictable. Here we report the discovery of a macrocyclic bis-thiourea derivative that catalyzes stereospecific invertive substitution pathways of glycosyl chlorides. The utility of the catalyst is demonstrated in the synthesis of trans-1,2-, cis-1,2-, and 2-deoxy-β-glycosides. Mechanistic studies are consistent with a cooperative mechanism in which an electrophile and a nucleophile are simultaneously activated to effect a stereospecific substitution reaction. Copyright © 2017, American Association for the Advancement of Science.

Laboratory procedures to generate viral metagenomes.

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Thurber, Rebecca V; Haynes, Matthew; Breitbart, Mya; Wegley, Linda; Rohwer, Forest

2009-01-01

This collection of laboratory protocols describes the steps to collect viruses from various samples with the specific aim of generating viral metagenome sequence libraries (viromes). Viral metagenomics, the study of uncultured viral nucleic acid sequences from different biomes, relies on several concentration, purification, extraction, sequencing and heuristic bioinformatic methods. No single technique can provide an all-inclusive approach, and therefore the protocols presented here will be discussed in terms of hypothetical projects. However, care must be taken to individualize each step depending on the source and type of viral-particles. This protocol is a description of the processes we have successfully used to: (i) concentrate viral particles from various types of samples, (ii) eliminate contaminating cells and free nucleic acids and (iii) extract, amplify and purify viral nucleic acids. Overall, a sample can be processed to isolate viral nucleic acids suitable for high-throughput sequencing in approximately 1 week.

O-GLYCOBASE version 4.0: a revised database of O-glycosylated proteins

DEFF Research Database (Denmark)

Gupta, Ramneek; Birch, Hanne; Rapacki, Krzysztof

1999-01-01

O-GLYCBASE is a database of glycoproteins with O-linked glycosylation sites. Entries with at least one experimentally verified O-glycosylation site have been complied from protein sequence databases and literature. Each entry contains information about the glycan involved, the species, sequence, ...

SEM visualization of glycosylated surface molecules using lectin-coated microspheres

Science.gov (United States)

Duke, J.; Janer, L.; Campbell, M.

1985-01-01

There are several techniques currently used to localize glycosylated surface molecules by scanning electron microscopy (Grinnell, 1980; Molday, 1976; Linthicum and Sell, 1975; Nicolson, 1974; Lo Buglio, et al, 1972). A simple and rapid method, using a modification of Grinnell's technique is reported here. Essentially, microspheres coated with Concavalin A are used to bind to glycosylated regions of the palatal shelf epithelium and are visualized in the scanning electron microscope (SEM).

Glycosylation of KSHV Encoded vGPCR Functions in Its Signaling and Tumorigenicity

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Hui Wu

2015-03-01

Full Text Available Kaposi’s sarcoma-associated herpesvirus (KSHV is a tumor virus and the etiologic agent of Kaposi’s Sarcoma (KS. KSHV G protein-coupled receptor (vGPCR is an oncogene that is implicated in malignancies associated with KHSV infection. In this study, we show that vGPCR undergoes extensive N-linked glycosylation within the extracellular domains, specifically asparagines 18, 22, 31 and 202. An immunofluorescence assay demonstrates that N-linked glycosylation are necessary for vGPCR trafficking to the cellular membrane. Employing vGPCR mutants whose glycosylation sites were ablated, we show that these vGPCR mutants failed to activate downstream signaling in cultured cells and were severely impaired to induce tumor formation in the xenograph nude mouse model. These findings support the conclusion that glycosylation is critical for vGPCR tumorigenesis and imply that chemokine regulation at the plasma membrane is crucial for vGPCR mediated signaling.

Bayesian mixture analysis for metagenomic community profiling.

Science.gov (United States)

Morfopoulou, Sofia; Plagnol, Vincent

2015-09-15

Deep sequencing of clinical samples is now an established tool for the detection of infectious pathogens, with direct medical applications. The large amount of data generated produces an opportunity to detect species even at very low levels, provided that computational tools can effectively profile the relevant metagenomic communities. Data interpretation is complicated by the fact that short sequencing reads can match multiple organisms and by the lack of completeness of existing databases, in particular for viral pathogens. Here we present metaMix, a Bayesian mixture model framework for resolving complex metagenomic mixtures. We show that the use of parallel Monte Carlo Markov chains for the exploration of the species space enables the identification of the set of species most likely to contribute to the mixture. We demonstrate the greater accuracy of metaMix compared with relevant methods, particularly for profiling complex communities consisting of several related species. We designed metaMix specifically for the analysis of deep transcriptome sequencing datasets, with a focus on viral pathogen detection; however, the principles are generally applicable to all types of metagenomic mixtures. metaMix is implemented as a user friendly R package, freely available on CRAN: http://cran.r-project.org/web/packages/metaMix sofia.morfopoulou.10@ucl.ac.uk Supplementary data are available at Bionformatics online. © The Author 2015. Published by Oxford University Press.

O-GLYCBASE: a revised database of O-glycosylated proteins

DEFF Research Database (Denmark)

Hansen, Jan; Lund, Ole; Nielsen, Jens O.

1996-01-01

O-GLYCBASE is a comprehensive database of information on glycoproteins and their O-linked glycosylation sites. Entries are compiled and revised from the SWISS-PROT and PIR databases as well as directly from recently published reports. Nineteen percent of the entries extracted from the databases n...... of mucin type O-glycosylation sites in mammalian glycoproteins exclusively from the primary sequence is made available by E-mail or WWW. The O-GLYCBASE database is also available electronically through our WWW server or by anonymous FTP....

Structural and Functional Consequences of Increased Tubulin Glycosylation in Diabetes Mellitus

Science.gov (United States)

Williams, Stuart K.; Howarth, Nancy L.; Devenny, James J.; Bitensky, Mark W.

1982-11-01

The extent of in vitro nonenzymatic glycosylation of purified rat brain tubulin was dependent on time and glucose concentration. Tubulin glycosylation profoundly inhibited GTP-dependent tubulin polymerization. Electron microscopy and NaDodSO4/polyacrylamide gel electrophoresis showed that glycosylated tubulin forms high molecular weight amorphous aggregates that are not disrupted by detergents or reducing agents. The amount of covalently bound NaB3H4-reducible sugars in tubulin recovered from brain of streptozotocin-induced diabetic rats was dramatically increased as compared with tubulin recovered from normal rat brain. Moreover, tubulin recovered from diabetic rat brain exhibited less GTP-induced polymerization than tubulin from nondiabetic controls. The possible implications of these data for diabetic neuropathy are discussed.

Validation of Metagenomic Next-Generation Sequencing Tests for Universal Pathogen Detection.

Science.gov (United States)

Schlaberg, Robert; Chiu, Charles Y; Miller, Steve; Procop, Gary W; Weinstock, George

2017-06-01

- Metagenomic sequencing can be used for detection of any pathogens using unbiased, shotgun next-generation sequencing (NGS), without the need for sequence-specific amplification. Proof-of-concept has been demonstrated in infectious disease outbreaks of unknown causes and in patients with suspected infections but negative results for conventional tests. Metagenomic NGS tests hold great promise to improve infectious disease diagnostics, especially in immunocompromised and critically ill patients. - To discuss challenges and provide example solutions for validating metagenomic pathogen detection tests in clinical laboratories. A summary of current regulatory requirements, largely based on prior guidance for NGS testing in constitutional genetics and oncology, is provided. - Examples from 2 separate validation studies are provided for steps from assay design, and validation of wet bench and bioinformatics protocols, to quality control and assurance. - Although laboratory and data analysis workflows are still complex, metagenomic NGS tests for infectious diseases are increasingly being validated in clinical laboratories. Many parallels exist to NGS tests in other fields. Nevertheless, specimen preparation, rapidly evolving data analysis algorithms, and incomplete reference sequence databases are idiosyncratic to the field of microbiology and often overlooked.

O-GLYCBASE version 3.0: a revised database of O-glycosylated proteins

DEFF Research Database (Denmark)

Hansen, Jan; Lund, Ole; Nilsson, Jette

1998-01-01

O-GLYCBASE is a revised database of information on glycoproteins and their O-linked glycosylation sites. Entries are compiled and revised from the literature, and from the sequence databases. Entries include informations about species, sequence, glycosylation sites and glycan type and is fully cr...

Biotechnological applications of functional metagenomics in the food and pharmaceutical industries.

Science.gov (United States)

Coughlan, Laura M; Cotter, Paul D; Hill, Colin; Alvarez-Ordóñez, Avelino

2015-01-01

Microorganisms are found throughout nature, thriving in a vast range of environmental conditions. The majority of them are unculturable or difficult to culture by traditional methods. Metagenomics enables the study of all microorganisms, regardless of whether they can be cultured or not, through the analysis of genomic data obtained directly from an environmental sample, providing knowledge of the species present, and allowing the extraction of information regarding the functionality of microbial communities in their natural habitat. Function-based screenings, following the cloning and expression of metagenomic DNA in a heterologous host, can be applied to the discovery of novel proteins of industrial interest encoded by the genes of previously inaccessible microorganisms. Functional metagenomics has considerable potential in the food and pharmaceutical industries, where it can, for instance, aid (i) the identification of enzymes with desirable technological properties, capable of catalyzing novel reactions or replacing existing chemically synthesized catalysts which may be difficult or expensive to produce, and able to work under a wide range of environmental conditions encountered in food and pharmaceutical processing cycles including extreme conditions of temperature, pH, osmolarity, etc; (ii) the discovery of novel bioactives including antimicrobials active against microorganisms of concern both in food and medical settings; (iii) the investigation of industrial and societal issues such as antibiotic resistance development. This review article summarizes the state-of-the-art functional metagenomic methods available and discusses the potential of functional metagenomic approaches to mine as yet unexplored environments to discover novel genes with biotechnological application in the food and pharmaceutical industries.

Biotechnological applications of functional metagenomics in the food and pharmaceutical industries

Directory of Open Access Journals (Sweden)

Laura M Coughlan

2015-06-01

Full Text Available Microorganisms are found throughout nature, thriving in a vast range of environmental conditions. The majority of them are unculturable or difficult to culture by traditional methods. Metagenomics enables the study of all microorganisms, regardless of whether they can be cultured or not, through the analysis of genomic data obtained directly from an environmental sample, providing knowledge of the species present and allowing the extraction of information regarding the functionality of microbial communities in their natural habitat. Function-based screenings, following the cloning and expression of metagenomic DNA in a heterologous host, can be applied to the discovery of novel proteins of industrial interest encoded by the genes of previously inaccessible microorganisms. Functional metagenomics has considerable potential in the food and pharmaceutical industries, where it can, for instance, aid (i the identification of enzymes with desirable technological properties, capable of catalysing novel reactions or replacing existing chemically synthesized catalysts which may be difficult or expensive to produce, and able to work under a wide range of environmental conditions encountered in food and pharmaceutical processing cycles including extreme conditions of temperature, pH, osmolarity, etc; (ii the discovery of novel bioactives including antimicrobials active against microorganisms of concern both in food and medical settings; (iii the investigation of industrial and societal issues such as antibiotic resistance development. This review article summarizes the state-of-the-art functional metagenomic methods available and discusses the potential of functional metagenomic approaches to mine as yet unexplored environments to discover novel genes with biotechnological application in the food and pharmaceutical industries.

Patterns of glycemic control using glycosylated hemoglobin in diabetics.

Science.gov (United States)

Kahlon, Arunpreet Singh; Pathak, Rambha

2011-07-01

Till now estimation of blood glucose is the highly effective method for diagnosing diabetes mellitus but it provides a short-term picture of control. More evidence is required to prove that plasma glucose and glycosylated hemoglobin levels together gives a better estimate of glycemic control and compliance with treatment. Indian diabetes risk score (IDRS) is a simplified screening tool for identifying undiagnosed diabetic subjects, requires minimum time, and effort and can help to considerably reduce the costs of screening. To study patterns of glycemic control using glycosylated hemoglobin in diabetic patients. To find out correlation between levels of plasma glucose and glycosylated hemoglobin in diabetics and to calculate IDRS of the study population. A cross sectional study was conducted among 300 known diabetic patients attending outpatient department of a rural medical college in Haryana, India. Following standard procedures and protocols FPG and glycosylated hemoglobin were measured to find out a pattern of glycemic control in them after taking their written and informed consent. A correlation between the levels of glycosylated hemoglobin and fasting blood glucose was also calculated. These patients were made to fill a performa and their demographic and clinical risk factors were noted and based on this, their IDRS was calculated. This was done to validate the IDRS in Indian rural population. Fifty-two percent of the population had fasting plasma glucose level between 125-150 mg/dl, 21% had this level between 151-175 mg/dl. Thirteen percent of the study subjects had HbA1C between 6.5-7.5, more than half (57.3%) had this value between 7.5-8.5, 12% and 18% had values between 8.5-9.5 and 9.5-10.5, respectively. Twelve percent of the participants had HbA1C level higher than 10.5. Correlation of fasting plasma glucose level and HbA1C was also studied and found that correlation coefficient came out to be .311. This correlation was found to be statistically

A highly abundant bacteriophage discovered in the unknown sequences of human faecal metagenomes

NARCIS (Netherlands)

Dutilh, Bas E; Cassman, Noriko; McNair, Katelyn; Sanchez, Savannah E; Silva, Genivaldo G Z; Boling, Lance; Barr, Jeremy J; Speth, Daan R; Seguritan, Victor; Aziz, Ramy K; Felts, Ben; Dinsdale, Elizabeth A; Mokili, John L; Edwards, Robert A

2014-01-01

Metagenomics, or sequencing of the genetic material from a complete microbial community, is a promising tool to discover novel microbes and viruses. Viral metagenomes typically contain many unknown sequences. Here we describe the discovery of a previously unidentified bacteriophage present in the

Diagnostic serum glycosylation profile in patients with intellectual disability as a result of MAN1B1 deficiency

DEFF Research Database (Denmark)

Van Scherpenzeel, Monique; Timal, Sharita; Rymen, Daisy

2014-01-01

Congenital disorders of glycosylation comprise a group of genetic defects with a high frequency of intellectual disability, caused by deficient glycosylation of proteins and lipids. The molecular basis of the majority of the congenital disorders of glycosylation type I subtypes, localized...... in the cytosol and endoplasmic reticulum, has been solved. However, elucidation of causative genes for defective Golgi glycosylation (congenital disorders of glycosylation type II) remains challenging because of a lack of sufficiently specific diagnostic serum methods. In a single patient with intellectual...... disability, whole-exome sequencing revealed MAN1B1 as congenital disorder of glycosylation type II candidate gene. A novel mass spectrometry method was applied for high-resolution glycoprofiling of intact plasma transferrin. A highly characteristic glycosylation signature was observed with hybrid type N...

Carbohydrates on Proteins: Site-Specific Glycosylation Analysis by Mass Spectrometry

Science.gov (United States)

Zhu, Zhikai; Desaire, Heather

2015-07-01

Glycosylation on proteins adds complexity and versatility to these biologically vital macromolecules. To unveil the structure-function relationship of glycoproteins, glycopeptide-centric analysis using mass spectrometry (MS) has become a method of choice because the glycan is preserved on the glycosylation site and site-specific glycosylation profiles of proteins can be readily determined. However, glycopeptide analysis is still challenging given that glycopeptides are usually low in abundance and relatively difficult to detect and the resulting data require expertise to analyze. Viewing the urgent need to address these challenges, emerging methods and techniques are being developed with the goal of analyzing glycopeptides in a sensitive, comprehensive, and high-throughput manner. In this review, we discuss recent advances in glycoprotein and glycopeptide analysis, with topics covering sample preparation, analytical separation, MS and tandem MS techniques, as well as data interpretation and automation.

Osteoblasts extracellular matrix induces vessel like structures through glycosylated collagen I

Energy Technology Data Exchange (ETDEWEB)

Palmieri, D. [Genetics, DIBIO, University of Genova, Corso Europa 26, 16132 Genova (Italy); Valli, M.; Viglio, S. [Department of Biochemistry, University of Pavia (Italy); Ferrari, N. [Istituto Nazionale per la ricerca sul Cancro, Genova (Italy); Ledda, B.; Volta, C. [Genetics, DIBIO, University of Genova, Corso Europa 26, 16132 Genova (Italy); Manduca, P., E-mail: man-via@unige.it [Genetics, DIBIO, University of Genova, Corso Europa 26, 16132 Genova (Italy)

2010-03-10

Extracellular matrix (ECM) plays a fundamental role in angiogenesis affecting endothelial cells proliferation, migration and differentiation. Vessels-like network formation in vitro is a reliable test to study the inductive effects of ECM on angiogenesis. Here we utilized matrix deposed by osteoblasts as substrate where the molecular and structural complexity of the endogenous ECM is preserved, to test if it induces vessel-like network formation by endothelial cells in vitro. ECM is more similar to the physiological substrate in vivo than other substrates previously utilized for these studies in vitro. Osteogenic ECM, prepared in vitro from mature osteoblasts at the phase of maximal deposition and glycosylation of collagen I, induces EAhy926, HUVEC, and HDMEC endothelial cells to form vessels-like structures and promotes the activation of metalloproteinase-2 (MMP-2); the functionality of the p-38/MAPK signaling pathway is required. Osteogenic ECM also induces a transient increase of CXCL12 and a decrease of the receptor CXCR4. The induction of vessel-like networks is dependent from proper glycosylation of collagens and does not occur on osteogenic ECMs if deglycosylated by -galactosidase or on less glycosylated ECMs derived from preosteoblasts and normal fibroblasts, while is sustained on ECM from osteogenesis imperfecta fibroblasts only when their mutation is associated with over-glycosylation of collagen type I. These data support that post-translational glycosylation has a role in the induction in endothelial cells in vitro of molecules conductive to self-organization in vessels-like structures.

Osteoblasts extracellular matrix induces vessel like structures through glycosylated collagen I

International Nuclear Information System (INIS)

Palmieri, D.; Valli, M.; Viglio, S.; Ferrari, N.; Ledda, B.; Volta, C.; Manduca, P.

2010-01-01

Extracellular matrix (ECM) plays a fundamental role in angiogenesis affecting endothelial cells proliferation, migration and differentiation. Vessels-like network formation in vitro is a reliable test to study the inductive effects of ECM on angiogenesis. Here we utilized matrix deposed by osteoblasts as substrate where the molecular and structural complexity of the endogenous ECM is preserved, to test if it induces vessel-like network formation by endothelial cells in vitro. ECM is more similar to the physiological substrate in vivo than other substrates previously utilized for these studies in vitro. Osteogenic ECM, prepared in vitro from mature osteoblasts at the phase of maximal deposition and glycosylation of collagen I, induces EAhy926, HUVEC, and HDMEC endothelial cells to form vessels-like structures and promotes the activation of metalloproteinase-2 (MMP-2); the functionality of the p-38/MAPK signaling pathway is required. Osteogenic ECM also induces a transient increase of CXCL12 and a decrease of the receptor CXCR4. The induction of vessel-like networks is dependent from proper glycosylation of collagens and does not occur on osteogenic ECMs if deglycosylated by -galactosidase or on less glycosylated ECMs derived from preosteoblasts and normal fibroblasts, while is sustained on ECM from osteogenesis imperfecta fibroblasts only when their mutation is associated with over-glycosylation of collagen type I. These data support that post-translational glycosylation has a role in the induction in endothelial cells in vitro of molecules conductive to self-organization in vessels-like structures.

Gene identification and protein classification in microbial metagenomic sequence data via incremental clustering

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Li Weizhong

2008-04-01

Full Text Available Abstract Background The identification and study of proteins from metagenomic datasets can shed light on the roles and interactions of the source organisms in their communities. However, metagenomic datasets are characterized by the presence of organisms with varying GC composition, codon usage biases etc., and consequently gene identification is challenging. The vast amount of sequence data also requires faster protein family classification tools. Results We present a computational improvement to a sequence clustering approach that we developed previously to identify and classify protein coding genes in large microbial metagenomic datasets. The clustering approach can be used to identify protein coding genes in prokaryotes, viruses, and intron-less eukaryotes. The computational improvement is based on an incremental clustering method that does not require the expensive all-against-all compute that was required by the original approach, while still preserving the remote homology detection capabilities. We present evaluations of the clustering approach in protein-coding gene identification and classification, and also present the results of updating the protein clusters from our previous work with recent genomic and metagenomic sequences. The clustering results are available via CAMERA, (http://camera.calit2.net. Conclusion The clustering paradigm is shown to be a very useful tool in the analysis of microbial metagenomic data. The incremental clustering method is shown to be much faster than the original approach in identifying genes, grouping sequences into existing protein families, and also identifying novel families that have multiple members in a metagenomic dataset. These clusters provide a basis for further studies of protein families.

Bio-Prospecting Laccases in the Bacterial Diversity of Activated Sludge From Pulp and Paper Industry.

Science.gov (United States)

Gupta, Vijaya; Capalash, Neena; Gupta, Naveen; Sharma, Prince

2017-03-01

Activated sludge is an artificial ecosystem known to harbor complex microbial communities. Bacterial diversity in activated sludge from pulp and paper industry was studied to bioprospect for laccase, the multicopper oxidase applicable in a large number of industries due to its ability to utilize a wide range of substrates. Bacterial diversity using 454 pyrosequencing and laccase diversity using degenerate primers specific to conserved copper binding domain of laccase like multicopper oxidase (LMCO) genes were investigated. 1231 OTUs out of 11,425 sequence reads for bacterial diversity and 11 OTUs out of 15 reads for LMCO diversity were formed. Phylum Proteobacteria (64.95 %) with genus Thauera (13.65 %) was most abundant followed by phylum Bacteriodetes (11.46 %) that included the dominant genera Paludibacter (1.93 %) and Lacibacter (1.32 %). In case of LMCOs, 40 % sequences showed affiliation with Proteobacteria and 46.6 % with unculturable bacteria, indicating considerable novelty, and 13.3 % with Bacteroidetes. LMCOs belonged to H and J families.

Fasting serum glucose and glycosylated hemoglobin level in obesity.

Science.gov (United States)

Das, R K; Nessa, A; Hossain, M A; Siddiqui, N I; Hussain, M A

2014-04-01

Obesity is a condition in which the body fat stores are increased to an extent which impairs health and leads to serious health consequences. The amount of body fat is difficult to measure directly, and is usually determined from an indirect measure - the body mass index (BMI). Increased BMI in obese persons is directly associated with an increase in metabolic disease, such as type 2 diabetes mellitus. This Analytical cross sectional study was undertaken to assess the relation between obesity and glycemic control of body by measuring fasting serum glucose and glycosylated hemoglobin. This study was carried out in the Department of Physiology, Mymensingh Medical College, Mymensingh from 1st July 2011 to 30th June 2012 on 120 equally divided male and female persons within the age range of 25 to 55 years. Age more than 55 years and less than 25 years and diagnosed case of Hypothyroidism, Cushing's syndrome, polycystic ovary, Antipsychotic drug user and regular steroid users were excluded. Non probability purposive type of sampling technique was used for selecting the study subjects. Measurement of body mass index was done as per procedure. Fasting serum glucose was estimated by glucose oxidase method and Glycosylated hemoglobin by Boronate Affinity method. Statistical analysis was done by SPSS (version 17.0). Data were expressed as Mean±SE and statistical significance of difference among the groups were calculated by unpaired student's 't' test and Pearson's correlation coefficient tests were done as applicable. The Mean±SE of fasting serum glucose was significant at 1% level (P value obese group of BMI. There was no significant difference of glycosylated hemoglobin level between control and study groups. But there was positive correlation within each group. Fasting serum glucose also showed a bit stronger positive correlation with BMI. Both obese male and female persons showed higher levels of fasting serum glucose and glycosylated hemoglobin. The observed positive

Involvement of Aberrant Glycosylation in Thyroid Cancer

Directory of Open Access Journals (Sweden)

Eiji Miyoshi

2010-01-01

Full Text Available Glycosylation is one of the most common posttranslational modification reactions and nearly half of all known proteins in eukaryotes are glycosylated. In fact, changes in oligosaccharides structures are associated with many physiological and pathological events, including cell growth, migration and differentiation, and tumor invasion. Therefore, functional glycomics, which is a comprehensive study of the structures and functions of glycans, is attracting the increasing attention of scientists in various fields of life science. In cases of thyroid cancer, the biological characters and prognosis are completely different in each type of histopathology, and their oligosaccharide structures as well as the expression of glycosyltransferases are also different. In this review, we summarized our previous papers on oligosaccharides and thyroid cancers and discussed a possible function of oligosaccharides in the carcinogenesis in thyroid cancer.

Extremozymes from metagenome: Potential applications in food processing.

Science.gov (United States)

Khan, Mahejibin; Sathya, T A

2017-06-12

The long-established use of enzymes for food processing and product formulation has resulted in an increased enzyme market compounding to 7.0% annual growth rate. Advancements in molecular biology and recognition that enzymes with specific properties have application for industrial production of infant, baby and functional foods boosted research toward sourcing the genes of microorganisms for enzymes with distinctive properties. In this regard, functional metagenomics for extremozymes has gained attention on the premise that such enzymes can catalyze specific reactions. Hence, metagenomics that can isolate functional genes of unculturable extremophilic microorganisms has expanded attention as a promising tool. Developments in this field of research in relation to food sector are reviewed.

Metagenomes provide valuable comparative information on soil microeukaryotes

DEFF Research Database (Denmark)

Jacquiod, Samuel Jehan Auguste; Stenbæk, Jonas; Santos, Susana

2016-01-01

has been identified. Our analyses suggest that publicly available metagenome data can provide valuable information on soil microeukaryotes for comparative purposes when handled appropriately, complementing the current view provided by ribosomal amplicon sequencing methods......., providing microbiologists with substantial amounts of accessible information. We took advantage of public metagenomes in order to investigate microeukaryote communities in a well characterized grassland soil. The data gathered allowed the evaluation of several factors impacting the community structure......, including the DNA extraction method, the database choice and also the annotation procedure. While most studies on soil microeukaryotes are based on sequencing of PCR-amplified taxonomic markers (18S rRNA genes, ITS regions), this work represents, to our knowledge, the first report based solely...

Multidimensional fractionation is a requirement for quantitation of Golgi-resident glycosylation enzymes from cultured human cells.

Science.gov (United States)

Lin, Chi-Hung; Chik, Jenny H L; Packer, Nicolle H; Molloy, Mark P

2015-02-06

Glycosylation results from the concerted action of glycosylation enzymes in the secretory pathway. In general, gene expression serves as the primary control mechanism, but post-translational fine-tuning of glycosylation enzyme functions is often necessary for efficient synthesis of specific glycan epitopes. While the field of glycomics has rapidly advanced, there lacks routine proteomic methods to measure expression of specific glycosylation enzymes needed to fill the gap between mRNA expression and the glycomic profile in a "reverse genomics" workflow. Toward developing this workflow we enriched Golgi membranes from two human colon cancer cell lines by sucrose density centrifugation and further mass-based fractionation by SDS-PAGE. We then applied mass spectrometry to demonstrate a doubling in the number of Golgi resident proteins identified, compared to the unenriched, low speed centrifuged supernatant of lysed cells. A total of 35 Golgi-resident glycosylation enzymes, of which 23 were glycosyltransferases, were identified making this the largest protein database so far of Golgi resident glycosylation enzymes experimentally identified in cultured human cells. We developed targeted mass spectrometry assays for specific quantitation of many of these glycosylation enzymes. Our results show that alterations in abundance of glycosylation enzymes at the protein level were generally consistent with the resultant glycomic profiles, but not necessarily with the corresponding glycosyltransferase mRNA expression as exemplified by the case of O-glycan core 1 T synthase.

Use of simulated data sets to evaluate the fidelity of metagenomic processing methods

Energy Technology Data Exchange (ETDEWEB)

Mavromatis, K [U.S. Department of Energy, Joint Genome Institute; Ivanova, N [U.S. Department of Energy, Joint Genome Institute; Barry, Kerrie [U.S. Department of Energy, Joint Genome Institute; Shapiro, Harris [U.S. Department of Energy, Joint Genome Institute; Goltsman, Eugene [U.S. Department of Energy, Joint Genome Institute; McHardy, Alice C. [IBM T. J. Watson Research Center; Rigoutsos, Isidore [IBM T. J. Watson Research Center; Salamov, Asaf [U.S. Department of Energy, Joint Genome Institute; Korzeniewski, Frank [U.S. Department of Energy, Joint Genome Institute; Land, Miriam L [ORNL; Lapidus, Alla L. [U.S. Department of Energy, Joint Genome Institute; Grigoriev, Igor [U.S. Department of Energy, Joint Genome Institute; Hugenholtz, Philip [U.S. Department of Energy, Joint Genome Institute; Kyrpides, Nikos C [U.S. Department of Energy, Joint Genome Institute

2007-01-01

Metagenomics is a rapidly emerging field of research for studying microbial communities. To evaluate methods presently used to process metagenomic sequences, we constructed three simulated data sets of varying complexity by combining sequencing reads randomly selected from 113 isolate genomes. These data sets were designed to model real metagenomes in terms of complexity and phylogenetic composition. We assembled sampled reads using three commonly used genome assemblers (Phrap, Arachne and JAZZ), and predicted genes using two popular gene-finding pipelines (fgenesb and CRITICA/GLIMMER). The phylogenetic origins of the assembled contigs were predicted using one sequence similarity-based ( blast hit distribution) and two sequence composition-based (PhyloPythia, oligonucleotide frequencies) binning methods. We explored the effects of the simulated community structure and method combinations on the fidelity of each processing step by comparison to the corresponding isolate genomes. The simulated data sets are available online to facilitate standardized benchmarking of tools for metagenomic analysis.

N-Glycosylation of Lipocalin 2 Is Not Required for Secretion or Exosome Targeting

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Erawan Borkham-Kamphorst

2018-04-01

Full Text Available Lipocalin 2 (LCN2 is a highly conserved secreted adipokine acting as a serum transport protein for small hydrophobic molecules such as fatty acids and steroids. In addition, LCN2 limits bacterial growth by sequestering iron-containing siderophores and further protects against intestinal inflammation and tumorigenesis associated with alterations in the microbiota. Human LCN2 contains one N-glycosylation site conserved in other species. It was postulated that this post-translational modification could facilitate protein folding, protects from proteolysis, is required for proper trafficking from the Golgi apparatus to the cell surface, and might be relevant for effective secretion. We here show that the homologous nucleoside antibiotic tunicamycin blocks N-linked glycosylation but not secretion of LCN2 in primary murine hepatocytes, derivatives thereof, human lung carcinoma cell line A549, and human prostate cancer cell line PC-3. Moreover, both the glycosylated and the non-glycosylated LCN2 variants are equally targeted to exosomes, demonstrating that this post-translational modification is not necessary for proper trafficking of LCN2 into these membranous extracellular vesicles. Furthermore, a hydrophobic cluster analysis revealed that the N-glycosylation site is embedded in a highly hydrophobic evolutionarily conserved surrounding. In sum, our data indicate that the N-glycosylation of LCN2 is not required for proper secretion and exosome cargo recruitment in different cell types, but might be relevant to increase overall solubility.

BeerDeCoded: the open beer metagenome project.

Science.gov (United States)

Sobel, Jonathan; Henry, Luc; Rotman, Nicolas; Rando, Gianpaolo

2017-01-01

Next generation sequencing has radically changed research in the life sciences, in both academic and corporate laboratories. The potential impact is tremendous, yet a majority of citizens have little or no understanding of the technological and ethical aspects of this widespread adoption. We designed BeerDeCoded as a pretext to discuss the societal issues related to genomic and metagenomic data with fellow citizens, while advancing scientific knowledge of the most popular beverage of all. In the spirit of citizen science, sample collection and DNA extraction were carried out with the participation of non-scientists in the community laboratory of Hackuarium, a not-for-profit organisation that supports unconventional research and promotes the public understanding of science. The dataset presented herein contains the targeted metagenomic profile of 39 bottled beers from 5 countries, based on internal transcribed spacer (ITS) sequencing of fungal species. A preliminary analysis reveals the presence of a large diversity of wild yeast species in commercial brews. With this project, we demonstrate that coupling simple laboratory procedures that can be carried out in a non-professional environment with state-of-the-art sequencing technologies and targeted metagenomic analyses, can lead to the detection and identification of the microbial content in bottled beer.

PhyloSift: phylogenetic analysis of genomes and metagenomes.

Science.gov (United States)

Darling, Aaron E; Jospin, Guillaume; Lowe, Eric; Matsen, Frederick A; Bik, Holly M; Eisen, Jonathan A

2014-01-01

Like all organisms on the planet, environmental microbes are subject to the forces of molecular evolution. Metagenomic sequencing provides a means to access the DNA sequence of uncultured microbes. By combining DNA sequencing of microbial communities with evolutionary modeling and phylogenetic analysis we might obtain new insights into microbiology and also provide a basis for practical tools such as forensic pathogen detection. In this work we present an approach to leverage phylogenetic analysis of metagenomic sequence data to conduct several types of analysis. First, we present a method to conduct phylogeny-driven Bayesian hypothesis tests for the presence of an organism in a sample. Second, we present a means to compare community structure across a collection of many samples and develop direct associations between the abundance of certain organisms and sample metadata. Third, we apply new tools to analyze the phylogenetic diversity of microbial communities and again demonstrate how this can be associated to sample metadata. These analyses are implemented in an open source software pipeline called PhyloSift. As a pipeline, PhyloSift incorporates several other programs including LAST, HMMER, and pplacer to automate phylogenetic analysis of protein coding and RNA sequences in metagenomic datasets generated by modern sequencing platforms (e.g., Illumina, 454).

PhyloSift: phylogenetic analysis of genomes and metagenomes

Directory of Open Access Journals (Sweden)

Aaron E. Darling

2014-01-01

Full Text Available Like all organisms on the planet, environmental microbes are subject to the forces of molecular evolution. Metagenomic sequencing provides a means to access the DNA sequence of uncultured microbes. By combining DNA sequencing of microbial communities with evolutionary modeling and phylogenetic analysis we might obtain new insights into microbiology and also provide a basis for practical tools such as forensic pathogen detection.In this work we present an approach to leverage phylogenetic analysis of metagenomic sequence data to conduct several types of analysis. First, we present a method to conduct phylogeny-driven Bayesian hypothesis tests for the presence of an organism in a sample. Second, we present a means to compare community structure across a collection of many samples and develop direct associations between the abundance of certain organisms and sample metadata. Third, we apply new tools to analyze the phylogenetic diversity of microbial communities and again demonstrate how this can be associated to sample metadata.These analyses are implemented in an open source software pipeline called PhyloSift. As a pipeline, PhyloSift incorporates several other programs including LAST, HMMER, and pplacer to automate phylogenetic analysis of protein coding and RNA sequences in metagenomic datasets generated by modern sequencing platforms (e.g., Illumina, 454.

The YNP metagenome project

DEFF Research Database (Denmark)

Inskeep, William P.; Jay, Zackary J.; Tringe, Susannah G.

2013-01-01

The Yellowstone geothermal complex contains over 10,000 diverse geothermal features that host numerous phylogenetically deeply rooted and poorly understood archaea, bacteria, and viruses. Microbial communities in high-temperature environments are generally less diverse than soil, marine, sediment......, and environmental variables. Twenty geochemically distinct geothermal ecosystems representing a broad spectrum of Yellowstone hot-spring environments were used for metagenomic and geochemical analysis and included approximately equal numbers of: (1) phototrophic mats, (2) “filamentous streamer” communities, and (3...

Tapping uncultured microorganisms through metagenomics for drug ...

African Journals Online (AJOL)

bdelnasser

reached the market using this new technology. For these reasons and others, the interest in natural products has ..... Functional metagenomic library screening strategy ..... Bertrand H, Poly F, Van VT, Lombard N, Nalin R, Vogel TM, Simonet P.

Glucosamine derived DISAL donors for stereoselective glycosylations under neutral conditions

DEFF Research Database (Denmark)

Grathe, S.; Thygesen, M.B.; Larsen, K.

2005-01-01

DISAL (methyl 3,5-dinitrosa/icylate) D-glcosyl, D-galactosyl, D-mannosyl, and L-quinovosyl donors have previously provided the efficient glycosylation of a range of substrates under either strictly neutral, mildly basic, or very mildly Lewis acidic (LiClO4) conditions. Herein we report the synthe......DISAL (methyl 3,5-dinitrosa/icylate) D-glcosyl, D-galactosyl, D-mannosyl, and L-quinovosyl donors have previously provided the efficient glycosylation of a range of substrates under either strictly neutral, mildly basic, or very mildly Lewis acidic (LiClO4) conditions. Herein we report...... the synthesis of new glucosamine DISAL donors, carrying N-TCP, -Troc, or -TFAc protecting groups, and their use in beta-(1,2-trans) selective glycosylations, primarily in NMP in the absence of any added Lewis acids, or in CH3NO2 with LiClO4. Finally, precise microwave heating proved effective in promoting...

Marked increase in rat red blood cell membrane protein glycosylation by one-month treatment with a cafeteria diet

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Laia Oliva

2015-07-01

Full Text Available Background and Objectives. Glucose, an aldose, spontaneously reacts with protein amino acids yielding glycosylated proteins. The compounds may reorganize to produce advanced glycosylation products, which regulatory importance is increasingly being recognized. Protein glycosylation is produced without the direct intervention of enzymes and results in the loss of function. Glycosylated plasma albumin, and glycosylated haemoglobin are currently used as index of mean plasma glucose levels, since higher glucose availability results in higher glycosylation rates. In this study we intended to detect the early changes in blood protein glycosylation elicited by an obesogenic diet.Experimental Design. Since albumin is in constant direct contact with plasma glucose, as are the red blood cell (RBC membranes, we analyzed their degree or glycosylation in female and male rats, either fed a standard diet or subjected to a hyper-energetic self-selected cafeteria diet for 30 days. This model produces a small increase in basal glycaemia and a significant increase in body fat, leaving the animals in the initial stages of development of metabolic syndrome. We also measured the degree of glycosylation of hemoglobin, and the concentration of glucose in contact with this protein, that within the RBC. Glycosylation was measured by colorimetric estimation of the hydroxymethylfurfural liberated from glycosyl residues by incubation with oxalate.Results. Plasma glucose was higher in cafeteria diet and in male rats, both independent effects. However, there were no significant differences induced by sex or diet in either hemoglobin or plasma proteins. Purified RBC membranes showed a marked effect of diet: higher glycosylation in cafeteria rats, which was more marked in females (not in controls. In any case, the number of glycosyl residues per molecule were higher in hemoglobin than in plasma proteins (after correction for molecular weight. The detected levels of glucose in

The binning of metagenomic contigs for microbial physiology of mixed cultures.

Science.gov (United States)

Strous, Marc; Kraft, Beate; Bisdorf, Regina; Tegetmeyer, Halina E

2012-01-01

So far, microbial physiology has dedicated itself mainly to pure cultures. In nature, cross feeding and competition are important aspects of microbial physiology and these can only be addressed by studying complete communities such as enrichment cultures. Metagenomic sequencing is a powerful tool to characterize such mixed cultures. In the analysis of metagenomic data, well established algorithms exist for the assembly of short reads into contigs and for the annotation of predicted genes. However, the binning of the assembled contigs or unassembled reads is still a major bottleneck and required to understand how the overall metabolism is partitioned over different community members. Binning consists of the clustering of contigs or reads that apparently originate from the same source population. In the present study eight metagenomic samples from the same habitat, a laboratory enrichment culture, were sequenced. Each sample contained 13-23 Mb of assembled contigs and up to eight abundant populations. Binning was attempted with existing methods but they were found to produce poor results, were slow, dependent on non-standard platforms or produced errors. A new binning procedure was developed based on multivariate statistics of tetranucleotide frequencies combined with the use of interpolated Markov models. Its performance was evaluated by comparison of the results between samples with BLAST and in comparison to existing algorithms for four publicly available metagenomes and one previously published artificial metagenome. The accuracy of the new approach was comparable or higher than existing methods. Further, it was up to a 100 times faster. It was implemented in Java Swing as a complete open source graphical binning application available for download and further development (http://sourceforge.net/projects/metawatt).

The binning of metagenomic contigs for microbial physiology of mixed cultures

Directory of Open Access Journals (Sweden)

Marc eStrous

2012-12-01

Full Text Available So far, microbial physiology has dedicated itself mainly to pure cultures. In nature, cross feeding and competition are important aspects of microbial physiology and these can only be addressed by studying complete communities such as enrichment cultures. Metagenomic sequencing is a powerful tool to characterize such mixed cultures. In the analysis of metagenomic data, well established algorithms exist for the assembly of short reads into contigs and for the annotation of predicted genes. However, the binning of the assembled contigs or unassembled reads is still a major bottleneck and required to understand how the overall metabolism is partitioned over different community members. Binning consists of the clustering of contigs or reads that apparently originate from the same source population.In the present study eight metagenomic samples originating from the same habitat, a laboratory enrichment culture, were sequenced. Each sample contained 13-23 Mb of assembled contigs and up to eight abundant populations. Binning was attempted with existing methods but they were found to produce poor results, were slow, dependent on non-standard platforms or produced errors. A new binning procedure was developed based on multivariate statistics of tetranucleotide frequencies combined with the use of interpolated Markov models. Its performance was evaluated by comparison of the results between samples with BLAST and in comparison to exisiting algorithms for four publicly available metagenomes and one previously published artificial metagenome. The accuracy of the new approach was comparable or higher than existing methods. Further, it was up to a hunderd times faster. It was implemented in Java Swing as a complete open source graphical binning application available for download and further development (http://sourceforge.net/projects/metawatt.

Mapping Sites of O-Glycosylation and Fringe Elongation on Drosophila Notch*

Science.gov (United States)

Harvey, Beth M.; Rana, Nadia A.; Moss, Hillary; Leonardi, Jessica; Jafar-Nejad, Hamed; Haltiwanger, Robert S.

2016-01-01

Glycosylation of the Notch receptor is essential for its activity and serves as an important modulator of signaling. Three major forms of O-glycosylation are predicted to occur at consensus sites within the epidermal growth factor-like repeats in the extracellular domain of the receptor: O-fucosylation, O-glucosylation, and O-GlcNAcylation. We have performed comprehensive mass spectral analyses of these three types of O-glycosylation on Drosophila Notch produced in S2 cells and identified peptides containing all 22 predicted O-fucose sites, all 18 predicted O-glucose sites, and all 18 putative O-GlcNAc sites. Using semiquantitative mass spectral methods, we have evaluated the occupancy and relative amounts of glycans at each site. The majority of the O-fucose sites were modified to high stoichiometries. Upon expression of the β3-N-acetylglucosaminyltransferase Fringe with Notch, we observed varying degrees of elongation beyond O-fucose monosaccharide, indicating that Fringe preferentially modifies certain sites more than others. Rumi modified O-glucose sites to high stoichiometries, although elongation of the O-glucose was site-specific. Although the current putative consensus sequence for O-GlcNAcylation predicts 18 O-GlcNAc sites on Notch, we only observed apparent O-GlcNAc modification at five sites. In addition, we performed mass spectral analysis on endogenous Notch purified from Drosophila embryos and found that the glycosylation states were similar to those found on Notch from S2 cells. These data provide foundational information for future studies investigating the mechanisms of how O-glycosylation regulates Notch activity. PMID:27268051

Diversity in protein glycosylation among insect species.

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Gianni Vandenborre

Full Text Available BACKGROUND: A very common protein modification in multicellular organisms is protein glycosylation or the addition of carbohydrate structures to the peptide backbone. Although the Class of the Insecta is the largest animal taxon on Earth, almost all information concerning glycosylation in insects is derived from studies with only one species, namely the fruit fly Drosophila melanogaster. METHODOLOGY/PRINCIPAL FINDINGS: In this report, the differences in glycoproteomes between insects belonging to several economically important insect orders were studied. Using GNA (Galanthus nivalis agglutinin affinity chromatography, different sets of glycoproteins with mannosyl-containing glycan structures were purified from the flour beetle (Tribolium castaneum, the silkworm (Bombyx mori, the honeybee (Apis mellifera, the fruit fly (D. melanogaster and the pea aphid (Acyrthosiphon pisum. To identify and characterize the purified glycoproteins, LC-MS/MS analysis was performed. For all insect species, it was demonstrated that glycoproteins were related to a broad range of biological processes and molecular functions. Moreover, the majority of glycoproteins retained on the GNA column were unique to one particular insect species and only a few glycoproteins were present in the five different glycoprotein sets. Furthermore, these data support the hypothesis that insect glycoproteins can be decorated with mannosylated O-glycans. CONCLUSIONS/SIGNIFICANCE: The results presented here demonstrate that oligomannose N-glycosylation events are highly specific depending on the insect species. In addition, we also demonstrated that protein O-mannosylation in insect species may occur more frequently than currently believed.

Metagenomics, metaMicrobesOnline and Kbase Data Integration (MICW - Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

Energy Technology Data Exchange (ETDEWEB)

Dehal, Paramvir

2011-10-12

Berkeley Lab's Paramvir Dehal on "Managing and Storing large Datasets in MicrobesOnline, metaMicrobesOnline and the DOE Knowledgebase" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

Patterns of glycemic control using glycosylated hemoglobin in diabetics

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Arunpreet Singh Kahlon

2011-01-01

Full Text Available Aim : Till now estimation of blood glucose is the highly effective method for diagnosing diabetes mellitus but it provides a short-term picture of control. More evidence is required to prove that plasma glucose and glycosylated hemoglobin levels together gives a better estimate of glycemic control and compliance with treatment. Indian diabetes risk score (IDRS is a simplified screening tool for identifying undiagnosed diabetic subjects, requires minimum time, and effort and can help to considerably reduce the costs of screening. Objective : To study patterns of glycemic control using glycosylated hemoglobin in diabetic patients. To find out correlation between levels of plasma glucose and glycosylated hemoglobin in diabetics and to calculate IDRS of the study population. Materials and Methods : A cross sectional study was conducted among 300 known diabetic patients attending outpatient department of a rural medical college in Haryana, India. Following standard procedures and protocols FPG and glycosylated hemoglobin were measured to find out a pattern of glycemic control in them after taking their written and informed consent. A correlation between the levels of glycosylated hemoglobin and fasting blood glucose was also calculated. These patients were made to fill a performa and their demographic and clinical risk factors were noted and based on this, their IDRS was calculated. This was done to validate the IDRS in Indian rural population. Results : Fifty-two percent of the population had fasting plasma glucose level between 125-150 mg/dl, 21% had this level between 151-175 mg/dl. Thirteen percent of the study subjects had HbA1C between 6.5-7.5, more than half (57.3% had this value between 7.5-8.5, 12% and 18% had values between 8.5-9.5 and 9.5-10.5, respectively. Twelve percent of the participants had HbA1C level higher than 10.5. Correlation of fasting plasma glucose level and HbA1C was also studied and found that correlation coefficient came

Metagenomics of the Svalbard reindeer rumen microbiome reveals abundance of polysaccharide utilization loci.

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Phillip B Pope

Full Text Available Lignocellulosic biomass remains a largely untapped source of renewable energy predominantly due to its recalcitrance and an incomplete understanding of how this is overcome in nature. We present here a compositional and comparative analysis of metagenomic data pertaining to a natural biomass-converting ecosystem adapted to austere arctic nutritional conditions, namely the rumen microbiome of Svalbard reindeer (Rangifer tarandus platyrhynchus. Community analysis showed that deeply-branched cellulolytic lineages affiliated to the Bacteroidetes and Firmicutes are dominant, whilst sequence binning methods facilitated the assemblage of metagenomic sequence for a dominant and novel Bacteroidales clade (SRM-1. Analysis of unassembled metagenomic sequence as well as metabolic reconstruction of SRM-1 revealed the presence of multiple polysaccharide utilization loci-like systems (PULs as well as members of more than 20 glycoside hydrolase and other carbohydrate-active enzyme families targeting various polysaccharides including cellulose, xylan and pectin. Functional screening of cloned metagenome fragments revealed high cellulolytic activity and an abundance of PULs that are rich in endoglucanases (GH5 but devoid of other common enzymes thought to be involved in cellulose degradation. Combining these results with known and partly re-evaluated metagenomic data strongly indicates that much like the human distal gut, the digestive system of herbivores harbours high numbers of deeply branched and as-yet uncultured members of the Bacteroidetes that depend on PUL-like systems for plant biomass degradation.

Endoplasmic reticulum stress and N-glycosylation modulate expression of WFS1 protein

International Nuclear Information System (INIS)

Yamaguchi, Suguru; Ishihara, Hisamitsu; Tamura, Akira; Yamada, Takahiro; Takahashi, Rui; Takei, Daisuke; Katagiri, Hideki; Oka, Yoshitomo

2004-01-01

Mutations of the WFS1 gene are responsible for two hereditary diseases, Wolfram syndrome and low frequency sensorineural hearing loss. The WFS1 protein is a glycoprotein located in the endoplasmic reticulum (ER) membrane but its function is poorly understood. Herein we show WFS1 mRNA and protein levels in pancreatic islets to be increased with ER-stress inducers, thapsigargin and dithiothreitol. Another ER-stress inducer, the N-glycosylation inhibitor tunicamycin, also raised WFS1 mRNA but not protein levels. Site-directed mutagenesis showed both Asn-663 and Asn-748 to be N-glycosylated in mouse WFS1 protein. The glycosylation-defective WFS1 protein, in which Asn-663 and Asn-748 had been substituted with aspartate, exhibited an increased protein turnover rate. Consistent with this, the WFS1 protein was more rapidly degraded in the presence of tunicamycin. These data indicate that ER-stress and N-glycosylation play important roles in WFS1 expression and stability, and also suggest regulatory roles for this protein in ER-stress induced cell death

Metagenomic species profiling using universal phylogenetic marker genes

DEFF Research Database (Denmark)

Sunagawa, Shinichi; Mende, Daniel R; Zeller, Georg

2013-01-01

To quantify known and unknown microorganisms at species-level resolution using shotgun sequencing data, we developed a method that establishes metagenomic operational taxonomic units (mOTUs) based on single-copy phylogenetic marker genes. Applied to 252 human fecal samples, the method revealed th...... that on average 43% of the species abundance and 58% of the richness cannot be captured by current reference genome-based methods. An implementation of the method is available at http://www.bork.embl.de/software/mOTU/.......To quantify known and unknown microorganisms at species-level resolution using shotgun sequencing data, we developed a method that establishes metagenomic operational taxonomic units (mOTUs) based on single-copy phylogenetic marker genes. Applied to 252 human fecal samples, the method revealed...

Challenges of the Unknown: Clinical Application of Microbial Metagenomics

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Graham Rose

2015-01-01

Full Text Available Availability of fast, high throughput and low cost whole genome sequencing holds great promise within public health microbiology, with applications ranging from outbreak detection and tracking transmission events to understanding the role played by microbial communities in health and disease. Within clinical metagenomics, identifying microorganisms from a complex and host enriched background remains a central computational challenge. As proof of principle, we sequenced two metagenomic samples, a known viral mixture of 25 human pathogens and an unknown complex biological model using benchtop technology. The datasets were then analysed using a bioinformatic pipeline developed around recent fast classification methods. A targeted approach was able to detect 20 of the viruses against a background of host contamination from multiple sources and bacterial contamination. An alternative untargeted identification method was highly correlated with these classifications, and over 1,600 species were identified when applied to the complex biological model, including several species captured at over 50% genome coverage. In summary, this study demonstrates the great potential of applying metagenomics within the clinical laboratory setting and that this can be achieved using infrastructure available to nondedicated sequencing centres.

Bioinformatic approaches reveal metagenomic characterization of soil microbial community.

Directory of Open Access Journals (Sweden)

Zhuofei Xu

Full Text Available As is well known, soil is a complex ecosystem harboring the most prokaryotic biodiversity on the Earth. In recent years, the advent of high-throughput sequencing techniques has greatly facilitated the progress of soil ecological studies. However, how to effectively understand the underlying biological features of large-scale sequencing data is a new challenge. In the present study, we used 33 publicly available metagenomes from diverse soil sites (i.e. grassland, forest soil, desert, Arctic soil, and mangrove sediment and integrated some state-of-the-art computational tools to explore the phylogenetic and functional characterizations of the microbial communities in soil. Microbial composition and metabolic potential in soils were comprehensively illustrated at the metagenomic level. A spectrum of metagenomic biomarkers containing 46 taxa and 33 metabolic modules were detected to be significantly differential that could be used as indicators to distinguish at least one of five soil communities. The co-occurrence associations between complex microbial compositions and functions were inferred by network-based approaches. Our results together with the established bioinformatic pipelines should provide a foundation for future research into the relation between soil biodiversity and ecosystem function.

Culture-independent discovery of natural products from soil metagenomes.

Science.gov (United States)

Katz, Micah; Hover, Bradley M; Brady, Sean F

2016-03-01

Bacterial natural products have proven to be invaluable starting points in the development of many currently used therapeutic agents. Unfortunately, traditional culture-based methods for natural product discovery have been deemphasized by pharmaceutical companies due in large part to high rediscovery rates. Culture-independent, or "metagenomic," methods, which rely on the heterologous expression of DNA extracted directly from environmental samples (eDNA), have the potential to provide access to metabolites encoded by a large fraction of the earth's microbial biosynthetic diversity. As soil is both ubiquitous and rich in bacterial diversity, it is an appealing starting point for culture-independent natural product discovery efforts. This review provides an overview of the history of soil metagenome-driven natural product discovery studies and elaborates on the recent development of new tools for sequence-based, high-throughput profiling of environmental samples used in discovering novel natural product biosynthetic gene clusters. We conclude with several examples of these new tools being employed to facilitate the recovery of novel secondary metabolite encoding gene clusters from soil metagenomes and the subsequent heterologous expression of these clusters to produce bioactive small molecules.

Introduction to Metagenomics at DOE JGI (Opening Remarks for the Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

Energy Technology Data Exchange (ETDEWEB)

Kyrpides, Nikos [DOE JGI

2011-10-12

After a quick introduction by DOE JGI Director Eddy Rubin, DOE JGI's Nikos Kyrpides delivers the opening remarks at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011

A broad pH range and processive chitinase from a metagenome library

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S.S. Thimoteo

Full Text Available Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked β(1-4 present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N′-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.

Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes.

Science.gov (United States)

Nielsen, H Bjørn; Almeida, Mathieu; Juncker, Agnieszka Sierakowska; Rasmussen, Simon; Li, Junhua; Sunagawa, Shinichi; Plichta, Damian R; Gautier, Laurent; Pedersen, Anders G; Le Chatelier, Emmanuelle; Pelletier, Eric; Bonde, Ida; Nielsen, Trine; Manichanh, Chaysavanh; Arumugam, Manimozhiyan; Batto, Jean-Michel; Quintanilha Dos Santos, Marcelo B; Blom, Nikolaj; Borruel, Natalia; Burgdorf, Kristoffer S; Boumezbeur, Fouad; Casellas, Francesc; Doré, Joël; Dworzynski, Piotr; Guarner, Francisco; Hansen, Torben; Hildebrand, Falk; Kaas, Rolf S; Kennedy, Sean; Kristiansen, Karsten; Kultima, Jens Roat; Léonard, Pierre; Levenez, Florence; Lund, Ole; Moumen, Bouziane; Le Paslier, Denis; Pons, Nicolas; Pedersen, Oluf; Prifti, Edi; Qin, Junjie; Raes, Jeroen; Sørensen, Søren; Tap, Julien; Tims, Sebastian; Ussery, David W; Yamada, Takuji; Renault, Pierre; Sicheritz-Ponten, Thomas; Bork, Peer; Wang, Jun; Brunak, Søren; Ehrlich, S Dusko

2014-08-01

Most current approaches for analyzing metagenomic data rely on comparisons to reference genomes, but the microbial diversity of many environments extends far beyond what is covered by reference databases. De novo segregation of complex metagenomic data into specific biological entities, such as particular bacterial strains or viruses, remains a largely unsolved problem. Here we present a method, based on binning co-abundant genes across a series of metagenomic samples, that enables comprehensive discovery of new microbial organisms, viruses and co-inherited genetic entities and aids assembly of microbial genomes without the need for reference sequences. We demonstrate the method on data from 396 human gut microbiome samples and identify 7,381 co-abundance gene groups (CAGs), including 741 metagenomic species (MGS). We use these to assemble 238 high-quality microbial genomes and identify affiliations between MGS and hundreds of viruses or genetic entities. Our method provides the means for comprehensive profiling of the diversity within complex metagenomic samples.

Mining the metagenome of activated biomass of an industrial wastewater treatment plant by a novel method.

Science.gov (United States)

Sharma, Nandita; Tanksale, Himgouri; Kapley, Atya; Purohit, Hemant J

2012-12-01

Metagenomic libraries herald the era of magnifying the microbial world, tapping into the vast metabolic potential of uncultivated microbes, and enhancing the rate of discovery of novel genes and pathways. In this paper, we describe a method that facilitates the extraction of metagenomic DNA from activated sludge of an industrial wastewater treatment plant and its use in mining the metagenome via library construction. The efficiency of this method was demonstrated by the large representation of the bacterial genome in the constructed metagenomic libraries and by the functional clones obtained. The BAC library represented 95.6 times the bacterial genome, while, the pUC library represented 41.7 times the bacterial genome. Twelve clones in the BAC library demonstrated lipolytic activity, while four clones demonstrated dioxygenase activity. Four clones in pUC library tested positive for cellulase activity. This method, using FTA cards, not only can be used for library construction, but can also store the metagenome at room temperature.

Potential and pitfalls of eukaryotic metagenome skimming: a test case for lichens.

Science.gov (United States)

Greshake, Bastian; Zehr, Simonida; Dal Grande, Francesco; Meiser, Anjuli; Schmitt, Imke; Ebersberger, Ingo

2016-03-01

Whole-genome shotgun sequencing of multispecies communities using only a single library layout is commonly used to assess taxonomic and functional diversity of microbial assemblages. Here, we investigate to what extent such metagenome skimming approaches are applicable for in-depth genomic characterizations of eukaryotic communities, for example lichens. We address how to best assemble a particular eukaryotic metagenome skimming data, what pitfalls can occur, and what genome quality can be expected from these data. To facilitate a project-specific benchmarking, we introduce the concept of twin sets, simulated data resembling the outcome of a particular metagenome sequencing study. We show that the quality of genome reconstructions depends essentially on assembler choice. Individual tools, including the metagenome assemblers Omega and MetaVelvet, are surprisingly sensitive to low and uneven coverages. In combination with the routine of assembly parameter choice to optimize the assembly N50 size, these tools can preclude an entire genome from the assembly. In contrast, MIRA, an all-purpose overlap assembler, and SPAdes, a multisized de Bruijn graph assembler, facilitate a comprehensive view on the individual genomes across a wide range of coverage ratios. Testing assemblers on a real-world metagenome skimming data from the lichen Lasallia pustulata demonstrates the applicability of twin sets for guiding method selection. Furthermore, it reveals that the assembly outcome for the photobiont Trebouxia sp. falls behind the a priori expectation given the simulations. Although the underlying reasons remain still unclear, this highlights that further studies on this organism require special attention during sequence data generation and downstream analysis. © 2015 John Wiley & Sons Ltd.

Novel polyhydroxyalkanoate copolymers produced in Pseudomonas putida by metagenomic polyhydroxyalkanoate synthases.

Science.gov (United States)

Cheng, Jiujun; Charles, Trevor C

2016-09-01

Bacterially produced biodegradable polyhydroxyalkanoates (PHAs) with versatile properties can be achieved using different PHA synthases (PhaCs). This work aims to expand the diversity of known PhaCs via functional metagenomics and demonstrates the use of these novel enzymes in PHA production. Complementation of a PHA synthesis-deficient Pseudomonas putida strain with a soil metagenomic cosmid library retrieved 27 clones expressing either class I, class II, or unclassified PHA synthases, and many did not have close sequence matches to known PhaCs. The composition of PHA produced by these clones was dependent on both the supplied growth substrates and the nature of the PHA synthase, with various combinations of short-chain-length (SCL) and medium-chain-length (MCL) PHA. These data demonstrate the ability to isolate diverse genes for PHA synthesis by functional metagenomics and their use for the production of a variety of PHA polymer and copolymer mixtures.

High-resolution metagenomics targets major functional types in complex microbial communities

Energy Technology Data Exchange (ETDEWEB)

Kalyuzhnaya, Marina G.; Lapidus, Alla; Ivanova, Natalia; Copeland, Alex C.; McHardy, Alice C.; Szeto, Ernest; Salamov, Asaf; Grigoriev, Igor V.; Suciu, Dominic; Levine, Samuel R.; Markowitz, Victor M.; Rigoutsos, Isidore; Tringe, Susannah G.; Bruce, David C.; Richardson, Paul M.; Lidstrom, Mary E.; Chistoserdova, Ludmila

2009-08-01

Most microbes in the biosphere remain uncultured and unknown. Whole genome shotgun (WGS) sequencing of environmental DNA (metagenomics) allows glimpses into genetic and metabolic potentials of natural microbial communities. However, in communities of high complexity metagenomics fail to link specific microbes to specific ecological functions. To overcome this limitation, we selectively targeted populations involved in oxidizing single-carbon (C{sub 1}) compounds in Lake Washington (Seattle, USA) by labeling their DNA via stable isotope probing (SIP), followed by WGS sequencing. Metagenome analysis demonstrated specific sequence enrichments in response to different C{sub 1} substrates, highlighting ecological roles of individual phylotypes. We further demonstrated the utility of our approach by extracting a nearly complete genome of a novel methylotroph Methylotenera mobilis, reconstructing its metabolism and conducting genome-wide analyses. This approach allowing high-resolution genomic analysis of ecologically relevant species has the potential to be applied to a wide variety of ecosystems.

Genome diversity of marine phages recovered from Mediterranean metagenomes: Size matters.

Directory of Open Access Journals (Sweden)

Mario López-Pérez

2017-09-01

Full Text Available Marine viruses play a critical role not only in the global geochemical cycles but also in the biology and evolution of their hosts. Despite their importance, viral diversity remains underexplored mostly due to sampling and cultivation challenges. Direct sequencing approaches such as viromics has provided new insights into the marine viral world. As a complementary approach, we analysed 24 microbial metagenomes (>0.2 μm size range obtained from six sites in the Mediterranean Sea that vary by depth, season and filter used to retrieve the fraction. Filter-size comparison showed a significant number of viral sequences that were retained on the larger-pore filters and were different from those found in the viral fraction from the same sample, indicating that some important viral information is missing using only assembly from viromes. Besides, we were able to describe 1,323 viral genomic fragments that were more than 10Kb in length, of which 36 represented complete viral genomes including some of them retrieved from a cross-assembly from different metagenomes. Host prediction based on sequence methods revealed new phage groups belonging to marine prokaryotes like SAR11, Cyanobacteria or SAR116. We also identified the first complete virophage from deep seawater and a new endemic clade of the recently discovered Marine group II Euryarchaeota virus. Furthermore, analysis of viral distribution using metagenomes and viromes indicated that most of the new phages were found exclusively in the Mediterranean Sea and some of them, mostly the ones recovered from deep metagenomes, do not recruit in any database probably indicating higher variability and endemicity in Mediterranean bathypelagic waters. Together these data provide the first detailed picture of genomic diversity, spatial and depth variations of viral communities within the Mediterranean Sea using metagenome assembly.

IN VITRO STUDY ON INHIBITION OF GLYCOSYLATION OF ...

African Journals Online (AJOL)

Administrator

complications of diabetes mellitus (Makita et al., 1991). Apart from protein ... enzymes; inhibition of regulatory molecule binding; crosslinking of glycosylated .... further investigation specific bio active compound responsible for such activities.

An integrated metagenome and -proteome analysis of the microbial community residing in a biogas production plant.

Science.gov (United States)

Ortseifen, Vera; Stolze, Yvonne; Maus, Irena; Sczyrba, Alexander; Bremges, Andreas; Albaum, Stefan P; Jaenicke, Sebastian; Fracowiak, Jochen; Pühler, Alfred; Schlüter, Andreas

2016-08-10

To study the metaproteome of a biogas-producing microbial community, fermentation samples were taken from an agricultural biogas plant for microbial cell and protein extraction and corresponding metagenome analyses. Based on metagenome sequence data, taxonomic community profiling was performed to elucidate the composition of bacterial and archaeal sub-communities. The community's cytosolic metaproteome was represented in a 2D-PAGE approach. Metaproteome databases for protein identification were compiled based on the assembled metagenome sequence dataset for the biogas plant analyzed and non-corresponding biogas metagenomes. Protein identification results revealed that the corresponding biogas protein database facilitated the highest identification rate followed by other biogas-specific databases, whereas common public databases yielded insufficient identification rates. Proteins of the biogas microbiome identified as highly abundant were assigned to the pathways involved in methanogenesis, transport and carbon metabolism. Moreover, the integrated metagenome/-proteome approach enabled the examination of genetic-context information for genes encoding identified proteins by studying neighboring genes on the corresponding contig. Exemplarily, this approach led to the identification of a Methanoculleus sp. contig encoding 16 methanogenesis-related gene products, three of which were also detected as abundant proteins within the community's metaproteome. Thus, metagenome contigs provide additional information on the genetic environment of identified abundant proteins. Copyright © 2016 Elsevier B.V. All rights reserved.

Ten years of maintaining and expanding a microbial genome and metagenome analysis system.

Science.gov (United States)

Markowitz, Victor M; Chen, I-Min A; Chu, Ken; Pati, Amrita; Ivanova, Natalia N; Kyrpides, Nikos C

2015-11-01

Launched in March 2005, the Integrated Microbial Genomes (IMG) system is a comprehensive data management system that supports multidimensional comparative analysis of genomic data. At the core of the IMG system is a data warehouse that contains genome and metagenome datasets sequenced at the Joint Genome Institute or provided by scientific users, as well as public genome datasets available at the National Center for Biotechnology Information Genbank sequence data archive. Genomes and metagenome datasets are processed using IMG's microbial genome and metagenome sequence data processing pipelines and are integrated into the data warehouse using IMG's data integration toolkits. Microbial genome and metagenome application specific data marts and user interfaces provide access to different subsets of IMG's data and analysis toolkits. This review article revisits IMG's original aims, highlights key milestones reached by the system during the past 10 years, and discusses the main challenges faced by a rapidly expanding system, in particular the complexity of maintaining such a system in an academic setting with limited budgets and computing and data management infrastructure. Copyright © 2015 Elsevier Ltd. All rights reserved.

Estimating DNA coverage and abundance in metagenomes using a gamma approximation

Energy Technology Data Exchange (ETDEWEB)

Hooper, Sean D; Dalevi, Daniel; Pati, Amrita; Mavromatis, Konstantinos; Ivanova, Natalia N; Kyrpides, Nikos C

2010-01-01

Shotgun sequencing generates large numbers of short DNA reads from either an isolated organism or, in the case of metagenomics projects, from the aggregate genome of a microbial community. These reads are then assembled based on overlapping sequences into larger, contiguous sequences (contigs). The feasibility of assembly and the coverage achieved (reads per nucleotide or distinct sequence of nucleotides) depend on several factors: the number of reads sequenced, the read length and the relative abundances of their source genomes in the microbial community. A low coverage suggests that most of the genomic DNA in the sample has not been sequenced, but it is often difficult to estimate either the extent of the uncaptured diversity or the amount of additional sequencing that would be most efficacious. In this work, we regard a metagenome as a population of DNA fragments (bins), each of which may be covered by one or more reads. We employ a gamma distribution to model this bin population due to its flexibility and ease of use. When a gamma approximation can be found that adequately fits the data, we may estimate the number of bins that were not sequenced and that could potentially be revealed by additional sequencing. We evaluated the performance of this model using simulated metagenomes and demonstrate its applicability on three recent metagenomic datasets.

Analysis and metabolic engineering of lipid-linked oligosaccharides in glycosylation-deficient CHO cells

International Nuclear Information System (INIS)

Jones, Meredith B.; Tomiya, Noboru; Betenbaugh, Michael J.; Krag, Sharon S.

2010-01-01

Glycosylation-deficient Chinese Hamster Ovary (CHO) cell lines can be used to expand our understanding of N-glycosylation pathways and to study Congenital Disorders of Glycosylation, diseases caused by defects in the synthesis of N-glycans. The mammalian N-glycosylation pathway involves the step-wise assembly of sugars onto a dolichol phosphate (P-Dol) carrier, forming a lipid-linked oligosaccharide (LLO), followed by the transfer of the completed oligosaccharide onto the protein of interest. In order to better understand how deficiencies in this pathway affect the availability of the completed LLO donor for use in N-glycosylation, we used a non-radioactive, HPLC-based assay to examine the intermediates in the LLO synthesis pathway for CHO-K1 cells and for three different glycosylation-deficient CHO cell lines. B4-2-1 cells, which have a mutation in the dolichol phosphate-mannose synthase (DPM2) gene, accumulated LLO with the structure Man 5 GlcNAc 2 -P-P-Dol, while MI8-5 cells, which lack glucosyltransferase I (ALG6) activity, accumulated Man 9 GlcNAc 2 -P-P-Dol. CHO-K1 and MI5-4 cells both produced primarily the complete LLO, Glc 3 Man 9 GlcNAc 2 -P-P-Dol, though the relative quantity was lower in MI5-4. MI5-4 cells have reduced hexokinase activity which could affect the availability of many of the substrates required for LLO synthesis and, consequently, impair production of the final LLO donor. Increasing hexokinase activity by overexpressing hexokinase II in MI5-4 caused a decrease in the relative quantities of the incomplete LLO intermediates from Man 5 GlcNAc 2 -PP-Dol through Glc 1 Man 9 GlcNAc 2 -PP-Dol, and an increase in the relative quantity of the final LLO donor, Glc 3 Man 9 GlcNAc 2 -P-P-Dol. This study suggests that metabolic engineering may be a useful strategy for improving LLO availability for use in N-glycosylation.

Analysis and metabolic engineering of lipid-linked oligosaccharides in glycosylation-deficient CHO cells

Energy Technology Data Exchange (ETDEWEB)

Jones, Meredith B., E-mail: mbauman7@jhu.edu [Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 North Charles Street, Maryland Hall 221, Baltimore, MD 21218 (United States); Tomiya, Noboru, E-mail: ntomiya1@jhu.edu [Department of Biology, Johns Hopkins University, 3400 North Charles Street, Mudd Hall 104A, Baltimore, MD 21218 (United States); Betenbaugh, Michael J., E-mail: beten@jhu.edu [Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 North Charles Street, Maryland Hall 221, Baltimore, MD 21218 (United States); Krag, Sharon S., E-mail: skrag@jhsph.edu [Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205 (United States)

2010-04-23

Glycosylation-deficient Chinese Hamster Ovary (CHO) cell lines can be used to expand our understanding of N-glycosylation pathways and to study Congenital Disorders of Glycosylation, diseases caused by defects in the synthesis of N-glycans. The mammalian N-glycosylation pathway involves the step-wise assembly of sugars onto a dolichol phosphate (P-Dol) carrier, forming a lipid-linked oligosaccharide (LLO), followed by the transfer of the completed oligosaccharide onto the protein of interest. In order to better understand how deficiencies in this pathway affect the availability of the completed LLO donor for use in N-glycosylation, we used a non-radioactive, HPLC-based assay to examine the intermediates in the LLO synthesis pathway for CHO-K1 cells and for three different glycosylation-deficient CHO cell lines. B4-2-1 cells, which have a mutation in the dolichol phosphate-mannose synthase (DPM2) gene, accumulated LLO with the structure Man{sub 5}GlcNAc{sub 2}-P-P-Dol, while MI8-5 cells, which lack glucosyltransferase I (ALG6) activity, accumulated Man{sub 9}GlcNAc{sub 2}-P-P-Dol. CHO-K1 and MI5-4 cells both produced primarily the complete LLO, Glc{sub 3}Man{sub 9}GlcNAc{sub 2}-P-P-Dol, though the relative quantity was lower in MI5-4. MI5-4 cells have reduced hexokinase activity which could affect the availability of many of the substrates required for LLO synthesis and, consequently, impair production of the final LLO donor. Increasing hexokinase activity by overexpressing hexokinase II in MI5-4 caused a decrease in the relative quantities of the incomplete LLO intermediates from Man{sub 5}GlcNAc{sub 2}-PP-Dol through Glc{sub 1}Man{sub 9}GlcNAc{sub 2}-PP-Dol, and an increase in the relative quantity of the final LLO donor, Glc{sub 3}Man{sub 9}GlcNAc{sub 2}-P-P-Dol. This study suggests that metabolic engineering may be a useful strategy for improving LLO availability for use in N-glycosylation.

Genome-scale metabolic model of Pichia pastoris with native and humanized glycosylation of recombinant proteins.

Science.gov (United States)

Irani, Zahra Azimzadeh; Kerkhoven, Eduard J; Shojaosadati, Seyed Abbas; Nielsen, Jens

2016-05-01

Pichia pastoris is used for commercial production of human therapeutic proteins, and genome-scale models of P. pastoris metabolism have been generated in the past to study the metabolism and associated protein production by this yeast. A major challenge with clinical usage of recombinant proteins produced by P. pastoris is the difference in N-glycosylation of proteins produced by humans and this yeast. However, through metabolic engineering, a P. pastoris strain capable of producing humanized N-glycosylated proteins was constructed. The current genome-scale models of P. pastoris do not address native nor humanized N-glycosylation, and we therefore developed ihGlycopastoris, an extension to the iLC915 model with both native and humanized N-glycosylation for recombinant protein production, but also an estimation of N-glycosylation of P. pastoris native proteins. This new model gives a better prediction of protein yield, demonstrates the effect of the different types of N-glycosylation of protein yield, and can be used to predict potential targets for strain improvement. The model represents a step towards a more complete description of protein production in P. pastoris, which is required for using these models to understand and optimize protein production processes. © 2015 Wiley Periodicals, Inc.

Metagenomic analysis of viral diversity in respiratory samples from patients with respiratory tract infections in Kuwait.

Science.gov (United States)

Madi, Nada; Al-Nakib, Widad; Mustafa, Abu Salim; Habibi, Nazima

2018-03-01

A metagenomic approach based on target independent next-generation sequencing has become a known method for the detection of both known and novel viruses in clinical samples. This study aimed to use the metagenomic sequencing approach to characterize the viral diversity in respiratory samples from patients with respiratory tract infections. We have investigated 86 respiratory samples received from various hospitals in Kuwait between 2015 and 2016 for the diagnosis of respiratory tract infections. A metagenomic approach using the next-generation sequencer to characterize viruses was used. According to the metagenomic analysis, an average of 145, 019 reads were identified, and 2% of these reads were of viral origin. Also, metagenomic analysis of the viral sequences revealed many known respiratory viruses, which were detected in 30.2% of the clinical samples. Also, sequences of non-respiratory viruses were detected in 14% of the clinical samples, while sequences of non-human viruses were detected in 55.8% of the clinical samples. The average genome coverage of the viruses was 12% with the highest genome coverage of 99.2% for respiratory syncytial virus, and the lowest was 1% for torque teno midi virus 2. Our results showed 47.7% agreement between multiplex Real-Time PCR and metagenomics sequencing in the detection of respiratory viruses in the clinical samples. Though there are some difficulties in using this method to clinical samples such as specimen quality, these observations are indicative of the promising utility of the metagenomic sequencing approach for the identification of respiratory viruses in patients with respiratory tract infections. © 2017 Wiley Periodicals, Inc.

Introduction to Metagenomics at DOE JGI: Program Overview and Program Informatics (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

Energy Technology Data Exchange (ETDEWEB)

Tringe, Susannah

2011-10-12

Susannah Tringe of the DOE Joint Genome Institute talks about the Program Overview and Program Informatics at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

Congenital disorders of glycosylation: The Saudi experience.

Science.gov (United States)

Alsubhi, Sarah; Alhashem, Amal; Faqeih, Eissa; Alfadhel, Majid; Alfaifi, Abdullah; Altuwaijri, Waleed; Alsahli, Saud; Aldhalaan, Hesham; Alkuraya, Fowzan S; Hundallah, Khalid; Mahmoud, Adel; Alasmari, Ali; Mutairi, Fuad Al; Abduraouf, Hanem; AlRasheed, Layan; Alshahwan, Saad; Tabarki, Brahim

2017-10-01

We retrospectively reviewed Saudi patients who had a congenital disorder of glycosylation (CDG). Twenty-seven Saudi patients (14 males, 13 females) from 13 unrelated families were identified. Based on molecular studies, the 27 CDG patients were classified into different subtypes: ALG9-CDG (8 patients, 29.5%), ALG3-CDG (7 patients, 26%), COG6-CDG (7 patients, 26%), MGAT2-CDG (3 patients, 11%), SLC35A2-CDG (1 patient), and PMM2-CDG (1 patient). All the patients had homozygous gene mutations. The combined carrier frequency of CDG for the encountered founder mutations in the Saudi population is 11.5 per 10,000, which translates to a minimum disease burden of 14 patients per 1,000,000. Our study provides comprehensive epidemiologic information and prevalence figures for each of these CDG in a large cohort of congenital disorder of glycosylation patients. © 2017 Wiley Periodicals, Inc.

Optimal Synthetic Glycosylation of a Therapeutic Antibody.

Science.gov (United States)

Parsons, Thomas B; Struwe, Weston B; Gault, Joseph; Yamamoto, Keisuke; Taylor, Thomas A; Raj, Ritu; Wals, Kim; Mohammed, Shabaz; Robinson, Carol V; Benesch, Justin L P; Davis, Benjamin G

2016-02-12

Glycosylation patterns in antibodies critically determine biological and physical properties but their precise control is a significant challenge in biology and biotechnology. We describe herein the optimization of an endoglycosidase-catalyzed glycosylation of the best-selling biotherapeutic Herceptin, an anti-HER2 antibody. Precise MS analysis of the intact four-chain Ab heteromultimer reveals nonspecific, non-enzymatic reactions (glycation), which are not detected under standard denaturing conditions. This competing reaction, which has hitherto been underestimated as a source of side products, can now be minimized. Optimization allowed access to the purest natural form of Herceptin to date (≥90 %). Moreover, through the use of a small library of sugars containing non-natural functional groups, Ab variants containing defined numbers of selectively addressable chemical tags (reaction handles at Sia C1) in specific positions (for attachment of cargo molecules or "glycorandomization") were readily generated.

Dengue Virus Glycosylation: What Do We Know?

Directory of Open Access Journals (Sweden)

Sally S. L. Yap

2017-07-01

Full Text Available In many infectious diseases caused by either viruses or bacteria, pathogen glycoproteins play important roles during the infection cycle, ranging from entry to successful intracellular replication and host immune evasion. Dengue is no exception. Dengue virus glycoproteins, envelope protein (E and non-structural protein 1 (NS1 are two popular sub-unit vaccine candidates. E protein on the virion surface is the major target of neutralizing antibodies. NS1 which is secreted during DENV infection has been shown to induce a variety of host responses through its binding to several host factors. However, despite their critical role in disease and protection, the glycosylated variants of these two proteins and their biological importance have remained understudied. In this review, we seek to provide a comprehensive summary of the current knowledge on protein glycosylation in DENV, and its role in virus biogenesis, host cell receptor interaction and disease pathogenesis.

Metagenomic approaches to exploit the biotechnological potential of the microbial consortia of marine sponges.

Science.gov (United States)

Kennedy, Jonathan; Marchesi, Julian R; Dobson, Alan D W

2007-05-01

Natural products isolated from sponges are an important source of new biologically active compounds. However, the development of these compounds into drugs has been held back by the difficulties in achieving a sustainable supply of these often-complex molecules for pre-clinical and clinical development. Increasing evidence implicates microbial symbionts as the source of many of these biologically active compounds, but the vast majority of the sponge microbial community remain uncultured. Metagenomics offers a biotechnological solution to this supply problem. Metagenomes of sponge microbial communities have been shown to contain genes and gene clusters typical for the biosynthesis of biologically active natural products. Heterologous expression approaches have also led to the isolation of secondary metabolism gene clusters from uncultured microbial symbionts of marine invertebrates and from soil metagenomic libraries. Combining a metagenomic approach with heterologous expression holds much promise for the sustainable exploitation of the chemical diversity present in the sponge microbial community.

Strain-Level Metagenomic Analysis of the Fermented Dairy Beverage Nunu Highlights Potential Food Safety Risks.

Science.gov (United States)

Walsh, Aaron M; Crispie, Fiona; Daari, Kareem; O'Sullivan, Orla; Martin, Jennifer C; Arthur, Cornelius T; Claesson, Marcus J; Scott, Karen P; Cotter, Paul D

2017-08-15

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. Copyright �

Conformationally superarmed S-ethyl glycosyl donors as effective building blocks for chemoselective oligosaccharide synthesis in one pot

DEFF Research Database (Denmark)

Bandara, Mithila D.; Yasomanee, Jagodige P.; Rath, Nigam P.

2017-01-01

A new series of superarmed glycosyl donors has been investigated. It was demonstrated that the S-ethyl leaving group allows for high reactivity, which is much higher than that of equally equipped S-phenyl glycosyl donors that were previously investigated by our groups. The superarmed S......-ethyl glycosyl donors equipped with a 2-O-benzoyl group gave complete β-stereoselectivity. Utility of the new glycosyl donors has been demonstrated in a one-pot one-addition oligosaccharide synthesis with all of the reaction components present from the beginning...

Low Density Lipoprotein Receptor Class A Repeats Are O-Glycosylated in Linker Regions

DEFF Research Database (Denmark)

Pedersen, Nis Borbye; Wang, Shengjun; Narimatsu, Yoshiki

2014-01-01

, which in wild-type CHO cells is glycosylated with the typical sialylated core 1 structure. The glycosites in linker regions of LDLR class A repeats are conserved in LDLR from man to Xenopus and found in other homologous receptors. O-Glycosylation is controlled by a large family of polypeptide Gal...

Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites

DEFF Research Database (Denmark)

Julenius, Karin; Mølgaard, Anne; Gupta, Ramneek

2005-01-01

could be predicted from averaged properties together with the fact that glycosylation sites are not precisely conserved indicates that mucin-type glycosylation in most cases is a bulk property and not a very site-specific one. NetOGlyc 3.1 is made available at www.cbs.dtu.dk/services/netoglyc....

Glycosylation as a Main Regulator of Growth and Death Factor Receptors Signaling

Directory of Open Access Journals (Sweden)

Inês Gomes Ferreira

2018-02-01

Full Text Available Glycosylation is a very frequent and functionally important post-translational protein modification that undergoes profound changes in cancer. Growth and death factor receptors and plasma membrane glycoproteins, which upon activation by extracellular ligands trigger a signal transduction cascade, are targets of several molecular anti-cancer drugs. In this review, we provide a thorough picture of the mechanisms bywhich glycosylation affects the activity of growth and death factor receptors in normal and pathological conditions. Glycosylation affects receptor activity through three non-mutually exclusive basic mechanisms: (1 by directly regulating intracellular transport, ligand binding, oligomerization and signaling of receptors; (2 through the binding of receptor carbohydrate structures to galectins, forming a lattice thatregulates receptor turnover on the plasma membrane; and (3 by receptor interaction with gangliosides inside membrane microdomains. Some carbohydrate chains, for example core fucose and β1,6-branching, exert a stimulatory effect on all receptors, while other structures exert opposite effects on different receptors or in different cellular contexts. In light of the crucial role played by glycosylation in the regulation of receptor activity, the development of next-generation drugs targeting glyco-epitopes of growth factor receptors should be considered a therapeutically interesting goal.

A novel cerebello-ocular syndrome with abnormal glycosylation due to abnormalities in dolichol metabolism.

NARCIS (Netherlands)

Morava, E.; Wevers, R.A.; Cantagrel, V.; Hoefsloot, L.H.; Al-Gazali, L.; Schoots, J.; Rooij, A. van; Huijben, K.; Ravenswaaij-Arts, C.M.A. van; Jongmans, M.C.J.; Sykut-Cegielska, J.; Hoffmann, G.F.; Bluemel, P.; Adamowicz, M.; Reeuwijk, J. van; Ng, B.G.; Bergman, J.E.; Bokhoven, J.H.L.M. van; Korner, C.; Babovic-Vuksanovic, D.; Willemsen, M.A.A.P.; Gleeson, J.G.; Lehle, L.; Brouwer, A.P.M. de; Lefeber, D.J.

2010-01-01

Cerebellar hypoplasia and slowly progressive ophthalmological symptoms are common features in patients with congenital disorders of glycosylation type I. In a group of patients with congenital disorders of glycosylation type I with unknown aetiology, we have previously described a distinct phenotype

Aberrant Glycosylation in the Left Ventricle and Plasma of Rats with Cardiac Hypertrophy and Heart Failure.

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Chiaki Nagai-Okatani

Full Text Available Targeted proteomics focusing on post-translational modifications, including glycosylation, is a useful strategy for discovering novel biomarkers. To apply this strategy effectively to cardiac hypertrophy and resultant heart failure, we aimed to characterize glycosylation profiles in the left ventricle and plasma of rats with cardiac hypertrophy. Dahl salt-sensitive hypertensive rats, a model of hypertension-induced cardiac hypertrophy, were fed a high-salt (8% NaCl diet starting at 6 weeks. As a result, they exhibited cardiac hypertrophy at 12 weeks and partially impaired cardiac function at 16 weeks compared with control rats fed a low-salt (0.3% NaCl diet. Gene expression analysis revealed significant changes in the expression of genes encoding glycosyltransferases and glycosidases. Glycoproteome profiling using lectin microarrays indicated upregulation of mucin-type O-glycosylation, especially disialyl-T, and downregulation of core fucosylation on N-glycans, detected by specific interactions with Amaranthus caudatus and Aspergillus oryzae lectins, respectively. Upregulation of plasma α-l-fucosidase activity was identified as a biomarker candidate for cardiac hypertrophy, which is expected to support the existing marker, atrial natriuretic peptide and its related peptides. Proteomic analysis identified cysteine and glycine-rich protein 3, a master regulator of cardiac muscle function, as an O-glycosylated protein with altered glycosylation in the rats with cardiac hypertrophy, suggesting that alternations in O-glycosylation affect its oligomerization and function. In conclusion, our data provide evidence of significant changes in glycosylation pattern, specifically mucin-type O-glycosylation and core defucosylation, in the pathogenesis of cardiac hypertrophy and heart failure, suggesting that they are potential biomarkers for these diseases.

Analysis of urinary PSA glycosylation is not indicative of high-risk prostate cancer.

Science.gov (United States)

Barrabés, Sílvia; Llop, Esther; Ferrer-Batallé, Montserrat; Ramírez, Manel; Aleixandre, Rosa N; Perry, Antoinette S; de Llorens, Rafael; Peracaula, Rosa

2017-07-01

The levels of core fucosylation and α2,3-linked sialic acid in serum Prostate Specific Antigen (PSA), using the lectins Pholiota squarrosa lectin (PhoSL) and Sambucus nigra agglutinin (SNA), can discriminate between Benign Prostatic Hyperplasia (BPH) and indolent prostate cancer (PCa) from aggressive PCa. In the present work we evaluated whether these glycosylation determinants could also be altered in urinary PSA obtained after digital rectal examination (DRE) and could also be useful for diagnosis determinations. For this purpose, α2,6-sialic acid and α1,6-fucose levels of urinary PSA from 53 patients, 18 biopsy-negative and 35 PCa patients of different aggressiveness degree, were analyzed by sandwich ELLA (Enzyme Linked Lectin Assay) using PhoSL and SNA. Changes in the levels of specific glycosylation determinants, that in serum PSA samples were indicative of PCa aggressiveness, were not found in PSA from DRE urine samples. Although urine is a simpler matrix for analyzing PSA glycosylation compared to serum, an immunopurification step was necessary to specifically detect the glycans on the PSA molecule. Those specific glycosylation determinants on urinary PSA were however not useful to improve PCa diagnosis. This could be probably due to the low proportion of PSA from the tumor in urine samples, which precludes the identification of aberrantly glycosylated PSA. Copyright © 2017 Elsevier B.V. All rights reserved.

NetOglyc: prediction of mucin type O-glycosylation sites based on sequence context and surface accessibility

DEFF Research Database (Denmark)

Hansen, Jan Erik; Lund, Ole; Tolstrup, Niels

1998-01-01

-glycosylated serine and threonine residues in independent test sets, thus proving more accurate than matrix statistics and vector projection methods. Predicition of O-glycosylation sites in the envelope glycoprotein gp120 from the primate lentiviruses HIV-1, HIV-2 and SIV are presented. The most conserved O...... structure and surface accessibility. The sequence context of glycosylated threonines was found to differ from that of serine, and the sites were found to cluster. Non-clustered sites had a sequence context different from that of clustered sites. charged residues were disfavoured at postition -1 and +3......-glycosylation signals in these evolutionary-related glycoproteins were found in their first hypervariable loop, V1. However, the strain variation for HIV-1 gp120 was significant. A computer server, available through WWW or E-mail, has been developed for prediction of mucin type O-glycosylation sites in proteins based...

Role of structure and glycosylation of adsorbed protein films in biolubrication.

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Deepak H Veeregowda

Full Text Available Water forms the basis of lubrication in the human body, but is unable to provide sufficient lubrication without additives. The importance of biolubrication becomes evident upon aging and disease, particularly under conditions that affect secretion or composition of body fluids. Insufficient biolubrication, may impede proper speech, mastication and swallowing, underlie excessive friction and wear of articulating cartilage surfaces in hips and knees, cause vaginal dryness, and result in dry, irritated eyes. Currently, our understanding of biolubrication is insufficient to design effective therapeutics to restore biolubrication. Aim of this study was to establish the role of structure and glycosylation of adsorbed protein films in biolubrication, taking the oral cavity as a model and making use of its dynamics with daily perturbations due to different glandular secretions, speech, drinking and eating, and tooth brushing. Using different surface analytical techniques (a quartz crystal microbalance with dissipation monitoring, colloidal probe atomic force microscopy, contact angle measurements and X-ray photo-electron spectroscopy, we demonstrated that adsorbed salivary conditioning films in vitro are more lubricious when their hydrophilicity and degree of glycosylation increase, meanwhile decreasing their structural softness. High-molecular-weight, glycosylated proteins adsorbing in loops and trains, are described as necessary scaffolds impeding removal of water during loading of articulating surfaces. Comparing in vitro and in vivo water contact angles measured intra-orally, these findings were extrapolated to the in vivo situation. Accordingly, lubricating properties of teeth, as perceived in 20 volunteers comprising of equal numbers of male and female subjects, could be related with structural softness and glycosylation of adsorbed protein films on tooth surfaces. Summarizing, biolubrication is due to a combination of structure and glycosylation

21 CFR 864.7470 - Glycosylated hemoglobin assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glycosylated hemoglobin assay. 864.7470 Section 864.7470 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7470...

Metagenomic analysis reveals presence of Treponema denticola in a tissue biopsy of the Iceman.

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Frank Maixner

Full Text Available Ancient hominoid genome studies can be regarded by definition as metagenomic analyses since they represent a mixture of both hominoid and microbial sequences in an environment. Here, we report the molecular detection of the oral spirochete Treponema denticola in ancient human tissue biopsies of the Iceman, a 5,300-year-old Copper Age natural ice mummy. Initially, the metagenomic data of the Iceman's genomic survey was screened for bacterial ribosomal RNA (rRNA specific reads. Through ranking the reads by abundance a relatively high number of rRNA reads most similar to T. denticola was detected. Mapping of the metagenome sequences against the T. denticola genome revealed additional reads most similar to this opportunistic pathogen. The DNA damage pattern of specifically mapped reads suggests an ancient origin of these sequences. The haematogenous spread of bacteria of the oral microbiome often reported in the recent literature could already explain the presence of metagenomic reads specific for T. denticola in the Iceman's bone biopsy. We extended, however, our survey to an Iceman gingival tissue sample and a mouth swab sample and could thereby detect T. denticola and Porphyrimonas gingivalis, another important member of the human commensal oral microflora. Taken together, this study clearly underlines the opportunity to detect disease-associated microorganisms when applying metagenomics-enabled approaches on datasets of ancient human remains.

A bioinformatics prediction approach towards analyzing the glycosylation, co-expression and interaction patterns of epithelial membrane antigen (EMA/MUC1)

International Nuclear Information System (INIS)

Kalra, Rajkumar S.; Wadhwa, Renu

2015-01-01

Epithelial membrane antigen (EMA or MUC1) is a heavily glycosylated, type I transmembrane glycoprotein commonly expressed by epithelial cells of duct organs. It has been shown to be aberrantly glycosylated in several diseases including cancer. Protein sequence based annotation and analysis of glycosylation profile of glycoproteins by robust computational and comprehensive algorithms provides possible insights to the mechanism(s) of anomalous glycosylation. In present report, by using a number of bioinformatics applications we studied EMA/MUC1 and explored its trans-membrane structural domain sequence that is widely subjected to glycosylation. Exploration of different extracellular motifs led to prediction of N and O-linked glycosylation target sites. Based on the putative O-linked target sites, glycosylated moieties and pathways were envisaged. Furthermore, Protein network analysis demonstrated physical interaction of EMA with a number of proteins and confirmed its functional involvement in cell growth and proliferation pathways. Gene Ontology analysis suggested an involvement of EMA in a number of functions including signal transduction, protein binding, processing and transport along with glycosylation. Thus, present study explored potential of bioinformatics prediction approach in analyzing glycosylation, co-expression and interaction patterns of EMA/MUC1 glycoprotein

A bioinformatics prediction approach towards analyzing the glycosylation, co-expression and interaction patterns of epithelial membrane antigen (EMA/MUC1)

Energy Technology Data Exchange (ETDEWEB)

Kalra, Rajkumar S., E-mail: renu-wadhwa@aist.go.jp; Wadhwa, Renu, E-mail: renu-wadhwa@aist.go.jp [Cell Proliferation Research Group and DBT-AIST International Laboratory for Advanced Biomedicine, National Institute of Advanced Industrial Science and Technology (AIST Central 4), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562 (Japan)

2015-02-27

Epithelial membrane antigen (EMA or MUC1) is a heavily glycosylated, type I transmembrane glycoprotein commonly expressed by epithelial cells of duct organs. It has been shown to be aberrantly glycosylated in several diseases including cancer. Protein sequence based annotation and analysis of glycosylation profile of glycoproteins by robust computational and comprehensive algorithms provides possible insights to the mechanism(s) of anomalous glycosylation. In present report, by using a number of bioinformatics applications we studied EMA/MUC1 and explored its trans-membrane structural domain sequence that is widely subjected to glycosylation. Exploration of different extracellular motifs led to prediction of N and O-linked glycosylation target sites. Based on the putative O-linked target sites, glycosylated moieties and pathways were envisaged. Furthermore, Protein network analysis demonstrated physical interaction of EMA with a number of proteins and confirmed its functional involvement in cell growth and proliferation pathways. Gene Ontology analysis suggested an involvement of EMA in a number of functions including signal transduction, protein binding, processing and transport along with glycosylation. Thus, present study explored potential of bioinformatics prediction approach in analyzing glycosylation, co-expression and interaction patterns of EMA/MUC1 glycoprotein.

Reduced apolipoprotein glycosylation in patients with the metabolic syndrome.

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Olga V Savinova

Full Text Available The purpose of this study was to compare the apolipoprotein composition of the three major lipoprotein classes in patients with metabolic syndrome to healthy controls.Very low density (VLDL, intermediate/low density (IDL/LDL, hereafter LDL, and high density lipoproteins (HDL fractions were isolated from plasma of 56 metabolic syndrome subjects and from 14 age-sex matched healthy volunteers. The apolipoprotein content of fractions was analyzed by one-dimensional (1D gel electrophoresis with confirmation by a combination of mass spectrometry and biochemical assays.Metabolic syndrome patients differed from healthy controls in the following ways: (1 total plasma--apoA1 was lower, whereas apoB, apoC2, apoC3, and apoE were higher; (2 VLDL--apoB, apoC3, and apoE were increased; (3 LDL--apoC3 was increased, (4 HDL--associated constitutive serum amyloid A protein (SAA4 was reduced (p<0.05 vs. controls for all. In patients with metabolic syndrome, the most extensively glycosylated (di-sialylated isoform of apoC3 was reduced in VLDL, LDL, and HDL fractions by 17%, 30%, and 25%, respectively (p<0.01 vs. controls for all. Similarly, the glycosylated isoform of apoE was reduced in VLDL, LDL, and HDL fractions by 15%, 26%, and 37% (p<0.01 vs. controls for all. Finally, glycosylated isoform of SAA4 in HDL fraction was 42% lower in patients with metabolic syndrome compared with controls (p<0.001.Patients with metabolic syndrome displayed several changes in plasma apolipoprotein composition consistent with hypertriglyceridemia and low HDL cholesterol levels. Reduced glycosylation of apoC3, apoE and SAA4 are novel findings, the pathophysiological consequences of which remain to be determined.

Glycosylation analysis of recombinant neutral protease I from Aspergillus oryzae expressed in Pichia pastoris.

Science.gov (United States)

Lei, Da; Xu, Yang; He, Qinghua; Pang, Yifeng; Chen, Bo; Xiong, Liang; Li, Yanping

2013-12-01

Neutral protease I from Aspergillus oryzae 3.042 was expressed in Pichia pastoris and its N-glycosylation properties were analyzed. After purification by nickel-affinity chromatography column, the recombinant neutral protease (rNPI) was confirmed to be N-glycosylated by periodicacid/Schiff's base staining and Endo H digestion. Moreover, the deglycosylated protein's molecular weight decreased to 43.3 kDa from 54.5 kDa analyzed by SDS-PAGE and MALDI-TOF-MS, and the hyperglycosylation extent was 21 %. The N-glycosylation site of rNPI was analyzed by nano LC-MS/MS after digesting by trypsin and Glu-C, and the unique potential site Asn41 of mature peptide was found to be glycosylated. Homology modeling of the 3D structure of rNPI indicated that the attached N-glycans hardly affected neutral protease's activity due to the great distance away from the active site of the enzyme.

The relative contribution of mannose salvage pathways to glycosylation in PMI-deficient mouse embryonic fibroblast cells.

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Fujita, Naonobu; Tamura, Ayako; Higashidani, Aya; Tonozuka, Takashi; Freeze, Hudson H; Nishikawa, Atsushi

2008-02-01

Mannose for mammalian glycan biosynthesis can be imported directly from the medium, derived from glucose or salvaged from endogenous or external glycans. All pathways must generate mannose 6-phosphate, the activated form of mannose. Imported or salvaged mannose is directly phosphorylated by hexokinase, whereas fructose 6-phosphate from glucose is converted to mannose 6-phosphate by phosphomannose isomerase (PMI). Normally, PMI provides the majority of mannose for glycan synthesis. To assess the contribution of PMI-independent pathways, we used PMI-null fibroblasts to study N-glycosylation of DNase I, a highly sensitive indicator protein. In PMI-null cells, imported mannose and salvaged mannose make a significant contribution to N-glycosylation. When these cells were grown in mannose-free medium along with the mannosidase inhibitor, swainsonine, to block the salvage pathways, N-glycosylation of DNase I was almost completely eliminated. Adding approximately 13 microm mannose to the medium completely restored normal glycosylation. Treatment with bafilomycin A(1), an inhibitor of lysosomal acidification, also markedly reduced N-glycosylation of DNase I, but in this case only 8 microm mannose was required to restore full glycosylation, indicating that a nonlysosomal source of mannose made a significant contribution. Glycosylation levels were greatly also reduced in glycoconjugate-free medium, when endosomal membrane trafficking was blocked by expression of a mutant SKD1. From these data, we conclude that PMI-null cells can salvage mannose from both endogenous and external glycoconjugates via lysosomal and nonlysosomal degradation pathways.

Experimental Design and Bioinformatics Analysis for the Application of Metagenomics in Environmental Sciences and Biotechnology.

Science.gov (United States)

Ju, Feng; Zhang, Tong

2015-11-03

Recent advances in DNA sequencing technologies have prompted the widespread application of metagenomics for the investigation of novel bioresources (e.g., industrial enzymes and bioactive molecules) and unknown biohazards (e.g., pathogens and antibiotic resistance genes) in natural and engineered microbial systems across multiple disciplines. This review discusses the rigorous experimental design and sample preparation in the context of applying metagenomics in environmental sciences and biotechnology. Moreover, this review summarizes the principles, methodologies, and state-of-the-art bioinformatics procedures, tools and database resources for metagenomics applications and discusses two popular strategies (analysis of unassembled reads versus assembled contigs/draft genomes) for quantitative or qualitative insights of microbial community structure and functions. Overall, this review aims to facilitate more extensive application of metagenomics in the investigation of uncultured microorganisms, novel enzymes, microbe-environment interactions, and biohazards in biotechnological applications where microbial communities are engineered for bioenergy production, wastewater treatment, and bioremediation.

MaxBin 2.0: an automated binning algorithm to recover genomes from multiple metagenomic datasets

Energy Technology Data Exchange (ETDEWEB)

Wu, Yu-Wei [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Simmons, Blake A. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Singer, Steven W. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

2015-10-29

The recovery of genomes from metagenomic datasets is a critical step to defining the functional roles of the underlying uncultivated populations. We previously developed MaxBin, an automated binning approach for high-throughput recovery of microbial genomes from metagenomes. Here, we present an expanded binning algorithm, MaxBin 2.0, which recovers genomes from co-assembly of a collection of metagenomic datasets. Tests on simulated datasets revealed that MaxBin 2.0 is highly accurate in recovering individual genomes, and the application of MaxBin 2.0 to several metagenomes from environmental samples demonstrated that it could achieve two complementary goals: recovering more bacterial genomes compared to binning a single sample as well as comparing the microbial community composition between different sampling environments. Availability and implementation: MaxBin 2.0 is freely available at http://sourceforge.net/projects/maxbin/ under BSD license. Supplementary information: Supplementary data are available at Bioinformatics online.

From cultured to uncultured genome sequences: metagenomics and modeling microbial ecosystems.

Science.gov (United States)

Garza, Daniel R; Dutilh, Bas E

2015-11-01

Microorganisms and the viruses that infect them are the most numerous biological entities on Earth and enclose its greatest biodiversity and genetic reservoir. With strength in their numbers, these microscopic organisms are major players in the cycles of energy and matter that sustain all life. Scientists have only scratched the surface of this vast microbial world through culture-dependent methods. Recent developments in generating metagenomes, large random samples of nucleic acid sequences isolated directly from the environment, are providing comprehensive portraits of the composition, structure, and functioning of microbial communities. Moreover, advances in metagenomic analysis have created the possibility of obtaining complete or nearly complete genome sequences from uncultured microorganisms, providing important means to study their biology, ecology, and evolution. Here we review some of the recent developments in the field of metagenomics, focusing on the discovery of genetic novelty and on methods for obtaining uncultured genome sequences, including through the recycling of previously published datasets. Moreover we discuss how metagenomics has become a core scientific tool to characterize eco-evolutionary patterns of microbial ecosystems, thus allowing us to simultaneously discover new microbes and study their natural communities. We conclude by discussing general guidelines and challenges for modeling the interactions between uncultured microorganisms and viruses based on the information contained in their genome sequences. These models will significantly advance our understanding of the functioning of microbial ecosystems and the roles of microbes in the environment.

Functional relevance of protein glycosylation to the pro-inflammatory effects of extracellular matrix metalloproteinase inducer (EMMPRIN) on monocytes/macrophages.

Science.gov (United States)

Ge, Heng; Yuan, Wei; Liu, Jidong; He, Qing; Ding, Song; Pu, Jun; He, Ben

2015-01-01

Extracellular matrix metalloproteinase inducer (EMMPRIN) is an important pro-inflammatory protein involved in the cellular functions of monocytes/macrophages. We have hypothesized that high-level heterogeneousness of protein glycosylation of EMMPRIN may have functional relevance to its biological effects and affect the inflammatory activity of monocytes/macrophages. The glycosylation patterns of EMMPRIN expressed by monocytes/macrophages (THP-1 cells) in response to different extracellular stimuli were observed, and the structures of different glycosylation forms were identified. After the purification of highly- and less-glycosylated proteins respectively, the impacts of different glycosylation forms on the pro-inflammatory effects of EMMPRIN were examined in various aspects, such as cell adhesion to endothelial cells, cell migrations, cytokine expression, and activation of inflammatory signalling pathway. 1) It was mainly the highly-glycosylated form of EMMPRIN (HG-EMMPRIN) that increased after being exposed to inflammatory signals (PMA and H2O2). 2) Glycosylation of EMMPRIN in monocytes/macrophages led to N-linked-glycans being added to the protein, with the HG form containing complex-type glycans and the less-glycosylated form (LG) the simple type. 3) Only the HG-EMMPRIN but not the LG-EMMPRIN exhibited pro-inflammatory effects and stimulated inflammatory activities of the monocytes/macrophages (i.e., activation of ERK1/2 and NF-κB pathway, enhanced monocyte-endothelium adhesion, cell migration and matrix metalloproteinase -9 expression). Post-transcriptional glycosylation represents an important mechanism that determines the biological effects of EMMPRIN in monocytes/macrophages. Glycosylation of EMMPRIN may serve as a potential target for regulating the inflammatory activities of monocytes/macrophages.

Functional relevance of protein glycosylation to the pro-inflammatory effects of extracellular matrix metalloproteinase inducer (EMMPRIN on monocytes/macrophages.

Directory of Open Access Journals (Sweden)

Heng Ge

Full Text Available Extracellular matrix metalloproteinase inducer (EMMPRIN is an important pro-inflammatory protein involved in the cellular functions of monocytes/macrophages. We have hypothesized that high-level heterogeneousness of protein glycosylation of EMMPRIN may have functional relevance to its biological effects and affect the inflammatory activity of monocytes/macrophages.The glycosylation patterns of EMMPRIN expressed by monocytes/macrophages (THP-1 cells in response to different extracellular stimuli were observed, and the structures of different glycosylation forms were identified. After the purification of highly- and less-glycosylated proteins respectively, the impacts of different glycosylation forms on the pro-inflammatory effects of EMMPRIN were examined in various aspects, such as cell adhesion to endothelial cells, cell migrations, cytokine expression, and activation of inflammatory signalling pathway.1 It was mainly the highly-glycosylated form of EMMPRIN (HG-EMMPRIN that increased after being exposed to inflammatory signals (PMA and H2O2. 2 Glycosylation of EMMPRIN in monocytes/macrophages led to N-linked-glycans being added to the protein, with the HG form containing complex-type glycans and the less-glycosylated form (LG the simple type. 3 Only the HG-EMMPRIN but not the LG-EMMPRIN exhibited pro-inflammatory effects and stimulated inflammatory activities of the monocytes/macrophages (i.e., activation of ERK1/2 and NF-κB pathway, enhanced monocyte-endothelium adhesion, cell migration and matrix metalloproteinase -9 expression.Post-transcriptional glycosylation represents an important mechanism that determines the biological effects of EMMPRIN in monocytes/macrophages. Glycosylation of EMMPRIN may serve as a potential target for regulating the inflammatory activities of monocytes/macrophages.

Beyond research: a primer for considerations on using viral metagenomics in the field and clinic

NARCIS (Netherlands)

Hall, Richard J; Draper, Jenny L; Nielsen, Fiona G G; Dutilh, Bas E

2015-01-01

Powered by recent advances in next-generation sequencing technologies, metagenomics has already unveiled vast microbial biodiversity in a range of environments, and is increasingly being applied in clinics for difficult-to-diagnose cases. It can be tempting to suggest that metagenomics could be used

Is glycosylated haemoglobin a marker of fertility?

DEFF Research Database (Denmark)

Hjollund, N H; Jensen, Tina Kold; Bonde, Jens Peter

1999-01-01

We performed a follow-up study of time to pregnancy in a population of first-time pregnancy planners without previous reproductive experience. The objective of this paper is to report and discuss a finding of a strong relationship between glycosylated haemoglobin (HbA1C) and fertility. A total...

Quantitative Field Testing Rotylenchulus reniformis DNA from Metagenomic Samples Isolated Directly from Soil

Science.gov (United States)

Showmaker, Kurt; Lawrence, Gary W.; Lu, Shien; Balbalian, Clarissa; Klink, Vincent P.

2011-01-01

A quantitative PCR procedure targeting the β-tubulin gene determined the number of Rotylenchulus reniformis Linford & Oliveira 1940 in metagenomic DNA samples isolated from soil. Of note, this outcome was in the presence of other soil-dwelling plant parasitic nematodes including its sister genus Helicotylenchus Steiner, 1945. The methodology provides a framework for molecular diagnostics of nematodes from metagenomic DNA isolated directly from soil. PMID:22194958

Glycosylation-Based Serum Biomarkers for Cancer Diagnostics and Prognostics.

Science.gov (United States)

Kirwan, Alan; Utratna, Marta; O'Dwyer, Michael E; Joshi, Lokesh; Kilcoyne, Michelle

2015-01-01

Cancer is the second most common cause of death in developed countries with approximately 14 million newly diagnosed individuals and over 6 million cancer-related deaths in 2012. Many cancers are discovered at a more advanced stage but better survival rates are correlated with earlier detection. Current clinically approved cancer biomarkers are most effective when applied to patients with widespread cancer. Single biomarkers with satisfactory sensitivity and specificity have not been identified for the most common cancers and some biomarkers are ineffective for the detection of early stage cancers. Thus, novel biomarkers with better diagnostic and prognostic performance are required. Aberrant protein glycosylation is well known hallmark of cancer and represents a promising source of potential biomarkers. Glycoproteins enter circulation from tissues or blood cells through active secretion or leakage and patient serum is an attractive option as a source for biomarkers from a clinical and diagnostic perspective. A plethora of technical approaches have been developed to address the challenges of glycosylation structure detection and determination. This review summarises currently utilised glycoprotein biomarkers and novel glycosylation-based biomarkers from the serum glycoproteome under investigation as cancer diagnostics and for monitoring and prognostics and includes details of recent high throughput and other emerging glycoanalytical techniques.

Abundance profiling of specific gene groups using precomputed gut metagenomes yields novel biological hypotheses.

Directory of Open Access Journals (Sweden)

Konstantin Yarygin

Full Text Available The gut microbiota is essentially a multifunctional bioreactor within a human being. The exploration of its enormous metabolic potential provides insights into the mechanisms underlying microbial ecology and interactions with the host. The data obtained using "shotgun" metagenomics capture information about the whole spectrum of microbial functions. However, each new study presenting new sequencing data tends to extract only a little of the information concerning the metabolic potential and often omits specific functions. A meta-analysis of the available data with an emphasis on biomedically relevant gene groups can unveil new global trends in the gut microbiota. As a step toward the reuse of metagenomic data, we developed a method for the quantitative profiling of user-defined groups of genes in human gut metagenomes. This method is based on the quick analysis of a gene coverage matrix obtained by pre-mapping the metagenomic reads to a global gut microbial catalogue. The method was applied to profile the abundance of several gene groups related to antibiotic resistance, phages, biosynthesis clusters and carbohydrate degradation in 784 metagenomes from healthy populations worldwide and patients with inflammatory bowel diseases and obesity. We discovered country-wise functional specifics in gut resistome and virome compositions. The most distinct features of the disease microbiota were found for Crohn's disease, followed by ulcerative colitis and obesity. Profiling of the genes belonging to crAssphage showed that its abundance varied across the world populations and was not associated with clinical status. We demonstrated temporal resilience of crAssphage and the influence of the sample preparation protocol on its detected abundance. Our approach offers a convenient method to add value to accumulated "shotgun" metagenomic data by helping researchers state and assess novel biological hypotheses.

O-GLYCBASE version 2.0: a revised database of O-glycosylated proteins

DEFF Research Database (Denmark)

Hansen, Jan; Lund, Ole; Rapacki, Kristoffer

1997-01-01

O-GLYCBASE is an updated database of information on glycoproteins and their O-linked glycosylation sites. Entries are compiled and revised from the literature, and from the SWISS-PROT database. Entries include information about species, sequence, glycosylation sites and glycan type. O-GLYCBASE is...... patterns for the GalNAc, mannose and GlcNAc transferases are shown. The O-GLYCBASE database is available through WWW or by anonymous FTP....

Characterization of kallikrein-related peptidase 4 glycosylations.

Science.gov (United States)

Yamakoshi, Yasuo; Yamakoshi, Fumiko; Hu, Jan C-C; Simmer, James P

2011-12-01

Kallikrein-related peptidase 4 (KLK4) is a glycosylated serine protease that functions in the maturation (hardening) of dental enamel. Pig and mouse KLK4 contain three potential N-glycosylation sites. We isolated KLK4 from developing pig and mouse molars and characterized their N-glycosylations. N-glycans were enzymatically released by digestion with N-glycosidase F and fluorescently labeled with 2-aminobenzoic acid. Normal-phase high-performance liquid chromatography (NP-HPLC) revealed N-glycans with no, or with one, two, or three sialic acid attachments in pig KLK4 and with no, or with one or two sialic acid attachments in mouse KLK4. The labeled N-glycans were digested with sialidase to generate the asialo N-glycan cores that were fractionated by reverse-phase HPLC, and their retention times were compared with similarly labeled glycan standards. The purified cores were characterized by mass spectrometric and monosaccharide composition analyses. We determined that pig and mouse KLK4 have NA2 and NA2F biantennary N-glycan cores. The pig triantennary core is NA3. The mouse triantennary core is NA3 with a fucose connected by an α1-6 linkage, indicating that it is attached to the first N-acetyglucosamine (NA3F). We conclude that pig KLK4 has NA2, NA2F, and NA3 N-glycan cores with no, or with one, two, or three sialic acids. Mouse KLK4 has NA2, NA2F, and NA3F N-glycan cores with no, or with one or two sialic acids. © 2011 Eur J Oral Sci.

Effects of N-glycosylation on protein conformation and dynamics: Protein Data Bank analysis and molecular dynamics simulation study.

Science.gov (United States)

Lee, Hui Sun; Qi, Yifei; Im, Wonpil

2015-03-09

N-linked glycosylation is one of the most important, chemically complex, and ubiquitous post-translational modifications in all eukaryotes. The N-glycans that are covalently linked to proteins are involved in numerous biological processes. There is considerable interest in developments of general approaches to predict the structural consequences of site-specific glycosylation and to understand how these effects can be exploited in protein design with advantageous properties. In this study, the impacts of N-glycans on protein structure and dynamics are systematically investigated using an integrated computational approach of the Protein Data Bank structure analysis and atomistic molecular dynamics simulations of glycosylated and deglycosylated proteins. Our study reveals that N-glycosylation does not induce significant changes in protein structure, but decreases protein dynamics, likely leading to an increase in protein stability. Overall, these results suggest not only a common role of glycosylation in proteins, but also a need for certain proteins to be properly glycosylated to gain their intrinsic dynamic properties.

A sampling and metagenomic sequencing-based methodology for monitoring antimicrobial resistance in swine herds

DEFF Research Database (Denmark)

Munk, Patrick; Dalhoff Andersen, Vibe; de Knegt, Leonardo

2016-01-01

Objectives Reliable methods for monitoring antimicrobial resistance (AMR) in livestock and other reservoirs are essential to understand the trends, transmission and importance of agricultural resistance. Quantification of AMR is mostly done using culture-based techniques, but metagenomic read...... mapping shows promise for quantitative resistance monitoring. Methods We evaluated the ability of: (i) MIC determination for Escherichia coli; (ii) cfu counting of E. coli; (iii) cfu counting of aerobic bacteria; and (iv) metagenomic shotgun sequencing to predict expected tetracycline resistance based...... cultivation-based techniques in terms of predicting expected tetracycline resistance based on antimicrobial consumption. Our metagenomic approach had sufficient resolution to detect antimicrobial-induced changes to individual resistance gene abundances. Pen floor manure samples were found to represent rectal...

MP3: a software tool for the prediction of pathogenic proteins in genomic and metagenomic data.

Science.gov (United States)

Gupta, Ankit; Kapil, Rohan; Dhakan, Darshan B; Sharma, Vineet K

2014-01-01

The identification of virulent proteins in any de-novo sequenced genome is useful in estimating its pathogenic ability and understanding the mechanism of pathogenesis. Similarly, the identification of such proteins could be valuable in comparing the metagenome of healthy and diseased individuals and estimating the proportion of pathogenic species. However, the common challenge in both the above tasks is the identification of virulent proteins since a significant proportion of genomic and metagenomic proteins are novel and yet unannotated. The currently available tools which carry out the identification of virulent proteins provide limited accuracy and cannot be used on large datasets. Therefore, we have developed an MP3 standalone tool and web server for the prediction of pathogenic proteins in both genomic and metagenomic datasets. MP3 is developed using an integrated Support Vector Machine (SVM) and Hidden Markov Model (HMM) approach to carry out highly fast, sensitive and accurate prediction of pathogenic proteins. It displayed Sensitivity, Specificity, MCC and accuracy values of 92%, 100%, 0.92 and 96%, respectively, on blind dataset constructed using complete proteins. On the two metagenomic blind datasets (Blind A: 51-100 amino acids and Blind B: 30-50 amino acids), it displayed Sensitivity, Specificity, MCC and accuracy values of 82.39%, 97.86%, 0.80 and 89.32% for Blind A and 71.60%, 94.48%, 0.67 and 81.86% for Blind B, respectively. In addition, the performance of MP3 was validated on selected bacterial genomic and real metagenomic datasets. To our knowledge, MP3 is the only program that specializes in fast and accurate identification of partial pathogenic proteins predicted from short (100-150 bp) metagenomic reads and also performs exceptionally well on complete protein sequences. MP3 is publicly available at http://metagenomics.iiserb.ac.in/mp3/index.php.

New Bacterial Phytase through Metagenomic Prospection

Directory of Open Access Journals (Sweden)

Nathálya Farias

2018-02-01

Full Text Available Alkaline phytases from uncultured microorganisms, which hydrolyze phytate to less phosphorylated myo-inositols and inorganic phosphate, have great potential as additives in agricultural industry. The development of metagenomics has stemmed from the ineluctable evidence that as-yet-uncultured microorganisms represent the vast majority of organisms in most environments on earth. In this study, a gene encoding a phytase was cloned from red rice crop residues and castor bean cake using a metagenomics strategy. The amino acid identity between this gene and its closest published counterparts is lower than 60%. The phytase was named PhyRC001 and was biochemically characterized. This recombinant protein showed activity on sodium phytate, indicating that PhyRC001 is a hydrolase enzyme. The enzymatic activity was optimal at a pH of 7.0 and at a temperature of 35 °C. β-propeller phytases possess great potential as feed additives because they are the only type of phytase with high activity at neutral pH. Therefore, to explore and exploit the underlying mechanism for β-propeller phytase functions could be of great benefit to biotechnology.

An enrichment of CRISPR and other defense-related features in marine sponge-associated microbial metagenomes

Directory of Open Access Journals (Sweden)

Hannes Horn

2016-11-01

Full Text Available Many marine sponges are populated by dense and taxonomically diverse microbial consortia. We employed a metagenomics approach to unravel the differences in the functional gene repertoire among three Mediterranean sponge species, Petrosia ficiformis, Sarcotragus foetidus, Aplysina aerophoba and seawater. Different signatures were observed between sponge and seawater metagenomes with regard to microbial community composition, GC content, and estimated bacterial genome size. Our analysis showed further a pronounced repertoire for defense systems in sponge metagenomes. Specifically, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR, restriction modification, DNA phosphorothioation and phage growth limitation systems were enriched in sponge metagenomes. These data suggest that defense is an important functional trait for an existence within sponges that requires mechanisms to defend against foreign DNA from microorganisms and viruses. This study contributes to an understanding of the evolutionary arms race between viruses/phages and bacterial genomes and it sheds light on the bacterial defenses that have evolved in the context of the sponge holobiont.

Metagenomic data of fungal internal transcribed spacer from serofluid dish, a traditional Chinese fermented food

Directory of Open Access Journals (Sweden)

Peng Chen

2016-03-01

Full Text Available Serofluid dish (or Jiangshui, in Chinese, a traditional food in the Chinese culture for thousands of years, is made from vegetables by fermentation. In this work, microorganism community of the fermented serofluid dish was investigated by the culture-independent method. The metagenomic data in this article contains the sequences of fungal internal transcribed spacer (ITS regions of rRNA genes from 12 different serofluid dish samples. The metagenome comprised of 50,865 average raw reads with an average of 8,958,220 bp and G + C content is 45.62%. This is the first report on metagenomic data of fungal ITS from serofluid dish employing Illumina platform to profile the fungal communities of this little known fermented food from Gansu Province, China. The Metagenomic data of fungal internal transcribed spacer can be accessed at NCBI, SRA database accession no. SRP067411. Keywords: Serofluid dish, Jiangshui, Fungal ITS, Cultivation-independent, Microbial diversity

Resolving prokaryotic taxonomy without rRNA: longer oligonucleotide word lengths improve genome and metagenome taxonomic classification.

Science.gov (United States)

Alsop, Eric B; Raymond, Jason

2013-01-01

Oligonucleotide signatures, especially tetranucleotide signatures, have been used as method for homology binning by exploiting an organism's inherent biases towards the use of specific oligonucleotide words. Tetranucleotide signatures have been especially useful in environmental metagenomics samples as many of these samples contain organisms from poorly classified phyla which cannot be easily identified using traditional homology methods, including NCBI BLAST. This study examines oligonucleotide signatures across 1,424 completed genomes from across the tree of life, substantially expanding upon previous work. A comprehensive analysis of mononucleotide through nonanucleotide word lengths suggests that longer word lengths substantially improve the classification of DNA fragments across a range of sizes of relevance to high throughput sequencing. We find that, at present, heptanucleotide signatures represent an optimal balance between prediction accuracy and computational time for resolving taxonomy using both genomic and metagenomic fragments. We directly compare the ability of tetranucleotide and heptanucleotide world lengths (tetranucleotide signatures are the current standard for oligonucleotide word usage analyses) for taxonomic binning of metagenome reads. We present evidence that heptanucleotide word lengths consistently provide more taxonomic resolving power, particularly in distinguishing between closely related organisms that are often present in metagenomic samples. This implies that longer oligonucleotide word lengths should replace tetranucleotide signatures for most analyses. Finally, we show that the application of longer word lengths to metagenomic datasets leads to more accurate taxonomic binning of DNA scaffolds and have the potential to substantially improve taxonomic assignment and assembly of metagenomic data.

Resolving prokaryotic taxonomy without rRNA: longer oligonucleotide word lengths improve genome and metagenome taxonomic classification.

Directory of Open Access Journals (Sweden)

Eric B Alsop

Full Text Available Oligonucleotide signatures, especially tetranucleotide signatures, have been used as method for homology binning by exploiting an organism's inherent biases towards the use of specific oligonucleotide words. Tetranucleotide signatures have been especially useful in environmental metagenomics samples as many of these samples contain organisms from poorly classified phyla which cannot be easily identified using traditional homology methods, including NCBI BLAST. This study examines oligonucleotide signatures across 1,424 completed genomes from across the tree of life, substantially expanding upon previous work. A comprehensive analysis of mononucleotide through nonanucleotide word lengths suggests that longer word lengths substantially improve the classification of DNA fragments across a range of sizes of relevance to high throughput sequencing. We find that, at present, heptanucleotide signatures represent an optimal balance between prediction accuracy and computational time for resolving taxonomy using both genomic and metagenomic fragments. We directly compare the ability of tetranucleotide and heptanucleotide world lengths (tetranucleotide signatures are the current standard for oligonucleotide word usage analyses for taxonomic binning of metagenome reads. We present evidence that heptanucleotide word lengths consistently provide more taxonomic resolving power, particularly in distinguishing between closely related organisms that are often present in metagenomic samples. This implies that longer oligonucleotide word lengths should replace tetranucleotide signatures for most analyses. Finally, we show that the application of longer word lengths to metagenomic datasets leads to more accurate taxonomic binning of DNA scaffolds and have the potential to substantially improve taxonomic assignment and assembly of metagenomic data.

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

Science.gov (United States)

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...

Thermotolerance and protein glycosylation: Inhibition studies with sodium fluoride, azauridine and tunicamycin

International Nuclear Information System (INIS)

Bursey, D.L.; Henle, K.J.; Nagle, W.A.; Moss, A.J.

1987-01-01

The glycosylation hypothesis predicts increased incorporation of monosaccharides into 0-linked glycoproteins during thermotolerance development and inhibition of thermotolerance when this process is blocked. Specific inhibitors of 0-linked glycosylation are not available. The authors examined the effect of non-specific inhibition of glycosylation on thermotolerance development by: 1. restriction of both exogenous sugars and endogeneous sugar synthesis with NaF to block glycolysis while providing L-glutamine as a substrate for ATP synthesis in the TCA cycle; or 2. inhibition of UDP-sugar synthesis using azauridine and tunicamycin. Inhibitors were added to cell cultures after heat conditioning (10 min, 45 0 ) and removed after 6 hr prior to 45 0 -test heating. Sugar deprivation was achieved with 10mM NaF in glucose-free EBSS, supplemented with 2mM L-glutamine. Synthesis of UDP-sugars was inhibited with 1mM azauridine + 1μg/ml tunicamycin. Thermotolerance development was inhibited 87% by NaF/glutamine and 47% by azauridine/tunicamycin. For example, the D/sub o/ of the thermotolerant cells was 42.5 min (control D/sub o/ = 3 min), but only 5.5 min with inhibition by the NaF solution. These results support the absolute requirement of sugar precursors for thermotolerance development as predicted by the glycosylation hypothesis

GLYCOSYLATED YGHJ POLYPEPTIDES FROM ENTEROTOXIGENIC ESCHERICHIA COLI (ETEC)

DEFF Research Database (Denmark)

2017-01-01

The present invention relates to glycosylated YghJ polypeptides from or derived from enterotoxigenic Escherichia coli (ETEC) that are immunogenic. In particular, the present invention relates to compositions or vaccines comprising the polypeptides and their application in immunization, vaccination...

MinION™ nanopore sequencing of environmental metagenomes: a synthetic approach.

Science.gov (United States)

Brown, Bonnie L; Watson, Mick; Minot, Samuel S; Rivera, Maria C; Franklin, Rima B

2017-03-01

Environmental metagenomic analysis is typically accomplished by assigning taxonomy and/or function from whole genome sequencing or 16S amplicon sequences. Both of these approaches are limited, however, by read length, among other technical and biological factors. A nanopore-based sequencing platform, MinION™, produces reads that are ≥1 × 104 bp in length, potentially providing for more precise assignment, thereby alleviating some of the limitations inherent in determining metagenome composition from short reads. We tested the ability of sequence data produced by MinION (R7.3 flow cells) to correctly assign taxonomy in single bacterial species runs and in three types of low-complexity synthetic communities: a mixture of DNA using equal mass from four species, a community with one relatively rare (1%) and three abundant (33% each) components, and a mixture of genomic DNA from 20 bacterial strains of staggered representation. Taxonomic composition of the low-complexity communities was assessed by analyzing the MinION sequence data with three different bioinformatic approaches: Kraken, MG-RAST, and One Codex. Results: Long read sequences generated from libraries prepared from single strains using the version 5 kit and chemistry, run on the original MinION device, yielded as few as 224 to as many as 3497 bidirectional high-quality (2D) reads with an average overall study length of 6000 bp. For the single-strain analyses, assignment of reads to the correct genus by different methods ranged from 53.1% to 99.5%, assignment to the correct species ranged from 23.9% to 99.5%, and the majority of misassigned reads were to closely related organisms. A synthetic metagenome sequenced with the same setup yielded 714 high quality 2D reads of approximately 5500 bp that were up to 98% correctly assigned to the species level. Synthetic metagenome MinION libraries generated using version 6 kit and chemistry yielded from 899 to 3497 2D reads with lengths averaging 5700 bp with up

A Systematic Study of Site-specific GalNAc-type O-Glycosylation Modulating Proprotein Convertase Processing

DEFF Research Database (Denmark)

Schjoldager, Katrine Ter-Borch Gram; Vester-Christensen, Malene B.; Goth, Christoffer K.

2011-01-01

Site-specific GalNAc-type O-glycosylation is emerging as an important co-regulator of proprotein convertase (PC) processing of proteins. PC processing is crucial in regulating many fundamental biological pathways and O-glycans in or immediately adjacent to processing sites may affect recognition...... and function of PCs. Thus, we previously demonstrated that deficiency in site-specific O-glycosylation in a PC site of the fibroblast growth factor, FGF23, resulted in marked reduction in secretion of active unprocessed FGF23, which cause familial tumoral calcinosis and hyperostosis hyperphosphatemia. GalNAc......-type O-glycosylation is found on serine and threonine amino acids and up to 20 distinct polypeptide GalNAc transferases catalyze the first addition of GalNAc to proteins making this step the most complex and differentially regulated steps in protein glycosylation. There is no reliable prediction model...

Metagenomic mining of feruloyl esterases from termite enteric flora

CSIR Research Space (South Africa)

Rashamuse, K

2014-01-01

Full Text Available A metagenome expression library was created from Trinervitermes trinervoides termite hindgut symbionts and subsequently screened for feruloyl esterase (FAE) activities, resulting in seven recombinant fosmids conferring feruloyl esterase phenotypes...

Comparative Metagenomics of Freshwater Microbial Communities

International Nuclear Information System (INIS)

Hemme, Chris; Deng, Ye; Tu, Qichao; Fields, Matthew; Gentry, Terry; Wu, Liyou; Tringe, Susannah; Watson, David; He, Zhili; Hazen, Terry; Tiedje, James; Rubin, Eddy; Zhou, Jizhong

2010-01-01

Previous analyses of a microbial metagenome from uranium and nitric-acid contaminated groundwater (FW106) showed significant environmental effects resulting from the rapid introduction of multiple contaminants. Effects include a massive loss of species and strain biodiversity, accumulation of toxin resistant genes in the metagenome and lateral transfer of toxin resistance genes between community members. To better understand these results in an ecological context, a second metagenome from a pristine groundwater system located along the same geological strike was sequenced and analyzed (FW301). It is hypothesized that FW301 approximates the ancestral FW106 community based on phylogenetic profiles and common geological parameters; however, even if is not the case, the datasets still permit comparisons between healthy and stressed groundwater ecosystems. Complex carbohydrate metabolism has been almost entirely lost in the stressed ecosystem. In contrast, the pristine system encodes a wide diversity of complex carbohydrate metabolism systems, suggesting that carbon turnover is very rapid and less leaky in the healthy groundwater system. FW301 encodes many (∼160+) carbon monoxide dehydrogenase genes while FW106 encodes none. This result suggests that the community is frequently exposed to oxygen from aerated rainwater percolating into the subsurface, with a resulting high rate of carbon metabolism and CO production. When oxygen levels fall, the CO then serves as a major carbon source for the community. FW301 appears to be capable of CO2 fixation via the reductive carboxylase (reverse TCA) cycle and possibly acetogenesis, activities; these activities are lacking in the heterotrophic FW106 system which relies exclusively on respiration of nitrate and/or oxygen for energy production. FW301 encodes a complete set of B12 biosynthesis pathway at high abundance suggesting the use of sodium gradients for energy production in the healthy groundwater community. Overall

fasting blood glucose and glycosylated haemoglobin levels

African Journals Online (AJOL)

Prince Acheampong

(HbA1c) levels of diabetes mellitus patients as an index of glycaemic control. It was a prospective case- finding study using laboratory and general practice records. ... range of glycosylated haemoglobins, and the cut-off values for some clinical .... quality of glycaemic control by glycated haemoglobin in out-patient diabetic ...

Proteomics and pathway analysis of N-glycosylated mammary gland proteins in response to Escherichia coli mastitis in cattle.

Science.gov (United States)

Yang, Yongxin; Shen, Weijun; Zhao, Xiaowei; Zhao, Huiling; Huang, Dongwei; Cheng, Guanglong

2014-06-01

The aim of this study was to investigate the N-linked glycosylated protein profile of mammary tissue from healthy cows and cows with mastitis due to Escherichia coli, in order to understand the molecular mechanisms of the host response to mastitis. N-glycopeptides were enriched with a lectin mixture and identified through high-accuracy mass spectrometry. A total of 551 N-glycosylation sites, corresponding to 294 proteins, were identified in the mammary tissues of healthy cows; these glycoproteins were categorised into three functional groups and clustered into 11 specific pathways. A total of 511 N-glycosylation sites, corresponding to 283 glycosylated proteins, were detected in the mammary tissues of cows with E. coli mastitis. There were differences in N-glycosylation sites in 98 proteins in the mammary tissues of healthy cows and cows with mastitis due to E. coli. Most proteins with altered glycosylation were those involved in responses to stress, cell adhesion and the immune response, and were assigned to five specific pathways based on their gene ontology annotation. The results from this study show that the glycosylated protein profile in the mammary tissues of healthy and mastitic cows are different, and altered glycoproteins are associated with several pathways, including the lysosome and O-glycan biosynthesis pathways. Copyright © 2014. Published by Elsevier Ltd.

A novel genome signature based on inter-nucleotide distances profiles for visualization of metagenomic data

Science.gov (United States)

Xie, Xian-Hua; Yu, Zu-Guo; Ma, Yuan-Lin; Han, Guo-Sheng; Anh, Vo

2017-09-01

There has been a growing interest in visualization of metagenomic data. The present study focuses on the visualization of metagenomic data using inter-nucleotide distances profile. We first convert the fragment sequences into inter-nucleotide distances profiles. Then we analyze these profiles by principal component analysis. Finally the principal components are used to obtain the 2-D scattered plot according to their source of species. We name our method as inter-nucleotide distances profiles (INP) method. Our method is evaluated on three benchmark data sets used in previous published papers. Our results demonstrate that the INP method is good, alternative and efficient for visualization of metagenomic data.

A compound heterozygous mutation in DPAGT1 results in a congenital disorder of glycosylation with a relatively mild phenotype

NARCIS (Netherlands)

Iqbal, Z.; Shahzad, M.; Vissers, L.E.L.M.; Scherpenzeel, M. van; Gilissen, C.; Razzaq, A.; Zahoor, M.Y.; Khan, S.N.; Kleefstra, T.; Veltman, J.A.; Brouwer, A.P.M. de; Lefeber, D.J.; Bokhoven, H. van; Riazuddin, S.

2013-01-01

Congenital disorders of glycosylation (CDG) are a large group of recessive multisystem disorders caused by impaired protein or lipid glycosylation. The CDG-I subgroup is characterized by protein N-glycosylation defects originating in the endoplasmic reticulum. The genetic defect is known for 17

Autotrophic microbe metagenomes and metabolic pathways differentiate adjacent red sea brine pools

KAUST Repository

Wang, Yong

2013-04-29

In the Red Sea, two neighboring deep-sea brine pools, Atlantis II and Discovery, have been studied extensively, and the results have shown that the temperature and concentrations of metal and methane in Atlantis II have increased over the past decades. Therefore, we investigated changes in the microbial community and metabolic pathways. Here, we compared the metagenomes of the two pools to each other and to those of deep-sea water samples. Archaea were generally absent in the Atlantis II metagenome; Bacteria in the metagenome were typically heterotrophic and depended on aromatic compounds and other extracellular organic carbon compounds as indicated by enrichment of the related metabolic pathways. In contrast, autotrophic Archaea capable of CO2 fixation and methane oxidation were identified in Discovery but not in Atlantis II. Our results suggest that hydrothermal conditions and metal precipitation in the Atlantis II pool have resulted in elimination of the autotrophic community and methanogens.

Metagenomic and proteomic analyses to elucidate the mechanism of anaerobic benzene degradation

Energy Technology Data Exchange (ETDEWEB)

Abu Laban, Nidal [Helmholtz (Germany)

2011-07-01

This paper presents the mechanism of anaerobic benzene degradation using metagenomic and proteomic analyses. The objective of the study is to find out the microbes and biochemistry involved in benzene degradation. Hypotheses are proposed for the initial activation mechanism of benzene under anaerobic conditions. Two methods for degradation, molecular characterization and identification of benzene-degrading enzymes, are described. The physiological and molecular characteristics of iron-reducing enrichment culture are given and the process is detailed. Metagenome analysis of iron-reducing culture is presented using a pie chart. From the metagenome analysis of benzene-degrading culture, putative mobile element genes were identified in the aromatic-degrading configurations. Metaproteomic analysis of iron-reducing cultures and the anaerobic benzene degradation pathway are also elucidated. From the study, it can be concluded that gram-positive bacteria are involved in benzene degradation under iron-reducing conditions and that the catalysis mechanism of putative anaerobic benzene carboxylase needs further investigation.

Metagenome reveals potential microbial degradation of hydrocarbon coupled with sulfate reduction in an oil-immersed chimney from Guaymas Basin

Directory of Open Access Journals (Sweden)

Ying eHe

2013-06-01

Full Text Available Deep-sea hydrothermal vent chimneys contain a high diversity of microorganisms, yet the metabolic activity and the ecological functions of the microbial communities remain largely unexplored. In this study, a metagenomic approach was applied to characterize the metabolic potential in a Guaymas hydrothermal vent chimney and to conduct comparative genomic analysis among a variety of environments with sequenced metagenomes. Complete clustering of functional gene categories with a comparative metagenomic approach showed that this Guaymas chimney metagenome was clustered most closely with a chimney metagenome from Juan de Fuca. All chimney samples were enriched with genes involved in recombination and repair, chemotaxis and flagellar assembly, highlighting their roles in coping with the fluctuating extreme deep-sea environments. A high proportion of transposases was observed in all the metagenomes from deep-sea chimneys, supporting the previous hypothesis that horizontal gene transfer may be common in the deep-sea vent chimney biosphere. In the Guaymas chimney metagenome, thermophilic sulfate reducing microorganisms including bacteria and archaea were found predominant, and genes coding for the degradation of refractory organic compounds such as cellulose, lipid, pullullan, as well as a few hydrocarbons including toluene, ethylbenzene and o-xylene were identified. Therefore, this oil-immersed chimney supported a thermophilic microbial community capable of oxidizing a range of hydrocarbons that served as electron donors for sulphate reduction under anaerobic conditions.

Modeling the mechanism of glycosylation reactions between ethanol, 1,2-ethanediol and methoxymethanol.

Science.gov (United States)

Azofra, Luis Miguel; Alkorta, Ibon; Toro-Labbé, Alejandro; Elguero, José

2013-09-07

The mechanism of the S(N)2 model glycosylation reaction between ethanol, 1,2-ethanediol and methoxymethanol has been studied theoretically at the B3LYP/6-311+G(d,p) computational level. Three different types of reactions have been explored: (i) the exchange of hydroxyl groups between these model systems; (ii) the basic catalysis reactions by combination of the substrates as glycosyl donors (neutral species) and acceptors (enolate species); and (iii) the effect on the reaction profile of an explicit H2O molecule in the reactions considered in (ii). The reaction force, the electronic chemical potential and the reaction electronic flux have been characterized for the reaction path in each case. Energy calculations show that methoxymethanol is the worst glycosyl donor model among the ones studied here, while 1,2-ethanediol is the best, having the lowest activation barrier of 74.7 kJ mol(-1) for the reaction between this one and the ethanolate as the glycosyl acceptor model. In general, the presence of direct interactions between the atoms involved in the penta-coordinated TS increases the activation energies of the processes.

Glycation and transglutaminase mediated glycosylation of fish gelatin peptides with glucosamine enhance bioactivity.

Science.gov (United States)

Hong, Pui Khoon; Gottardi, Davide; Ndagijimana, Maurice; Betti, Mirko

2014-01-01

A mixture of novel glycopeptides from glycosylation between cold water fish skin gelatin hydrolysates and glucosamine (GlcN) via transglutaminase (TGase), as well as glycation between fish gelatin hydrolysate and GlcN were identified by their pattern of molecular distribution using MALDI-TOF-MS. Glycated/glycosylated hydrolysates showed superior bioactivity to their original hydrolysates. Alcalase-derived fish skin gelatin hydrolysate glycosylated with GlcN in the presence of TGase at 25°C (FAT25) possessed antioxidant activity when tested in a linoleic acid oxidation system, when measured according to its 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity and when tested at the cellular level with human hepatocarcinoma (HepG2) cells as target cells. In addition, Alcalase-derived glycosylated hydrolysates showed specificity toward the inhibition of Escherichia coli (E. coli). The Flavourzyme-derived glycopeptides prepared at 37°C (FFC37 and FFT37) showed better DPPH scavenging activity than their native hydrolysates. The glycated Flavourzyme-derived hydrolysates were found to act as potential antimicrobial agents when incubated with E. coli and Bacillus subtilis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Comparative Glycoproteome Analysis: Dynamics of Protein Glycosylation during Metamorphic Transition from Pelagic to Benthic Life Stages in Three Invertebrates

KAUST Repository

Chandramouli, Kondethimmanahalli

2012-02-03

The life cycle of most benthic marine invertebrates has two distinct stages: the pelagic larval stage and the sessile juvenile stage. The transition between the larval stage and the juvenile stage is often abrupt and may be triggered by post-translational modification of proteins. Glycosylation, a very important post-translational modification, influences the biological activity of proteins. We used two-dimensional gel electrophoresis (2-DE) followed by glycoprotein-specific fluorescence staining and mass spectrometry with the goal of identifying glycosylation pattern changes during larval settlement and metamorphosis in barnacles, bryozoans, and polychaetes. Our results revealed substantial changes in the protein glycosylation patterns from larval to juvenile stages. Before metamorphosis, the degree of protein glycosylation was high in the barnacle Balanus (=Amphibalanus) amphitrite and the spionid polychaete Pseudopolydora vexillosa, whereas it increased after metamorphosis in the bryozoan Bugula neritina. We identified 19 abundant and differentially glycosylated proteins in these three species. Among the proteins, cellular stress- and metabolism-related proteins exhibited distinct glycosylation in B. amphitrite and B. neritina, whereas fatty acid metabolism-related proteins were abundantly glycosylated in P. vexillosa. Furthermore, the protein and gene expression analysis of some selected glycoproteins revealed that the degree of protein glycosylation did not always complement with transcriptional and translational changes associated with the larval-juvenile transition. The current study provides preliminary information on protein glycosylation in marine invertebrates that will serve as a solid basis for future comprehensive analysis of glycobiology during larval settlement and metamorphosis. © 2011 American Chemical Society.

Fab glycosylation of immunoglobulin G does not associate with improvement of rheumatoid arthritis during pregnancy

NARCIS (Netherlands)

A. Bondt (Albert); M. Wuhrer (Manfred); T.M. Kuijper (Martijn); J.M.W. Hazes (Mieke); R.J.E.M. Dolhain (Radboud)

2016-01-01

textabstractBackground: Changes in immunoglobulin G (IgG) constant domain (Fc) glycosylation are associated with changes in rheumatoid arthritis (RA) disease activity in response to pregnancy. Here, we sought to determine whether the same holds true for variable domain (Fab) glycosylation. Methods:

The glycosylated IgII extracellular domain of EMMPRIN is implicated in the induction of MMP-2.

Science.gov (United States)

Papadimitropoulou, Adriana; Mamalaki, Avgi

2013-07-01

EMMPRIN is a widely expressed transmembrane glycoprotein that plays important roles in many physiological and pathological processes, such as tumor invasion and metastasis. It stimulates the production of matrix metalloproteinase (MMPs) by tumor-associated fibroblasts. In the present study, our aim was to (a) to investigate if the IgII loop domain of the extracellular domain (ECD) of EMMPRIN contributes to the MMP production by fibroblasts and (b) to evaluate the significance of glycosylation in this process. For this purpose, we expressed the ECD, IgI, or IgII domains of EMMPRIN, in their glycosylated and non-glycosylated forms, in the heterologous expression systems of P. pastoris and E. coli, respectively. Dermal fibroblasts were treated with purified recombinant domains and proteins from cell extracts and supernatants were analyzed by Western blot and zymography assays. Fibroblasts treated with ECD-, IgI-, and IgII-glycosylated domains of EMMPRIN significantly stimulated the gelatinolytic activity of MMP-2, compared to untreated fibroblasts, whereas no significant effect was observed after treatment with the non-glycosylated ECD, IgI, and IgII domains. Western blot analysis from cell extracts and supernatants revealed that only the glycosylated forms were able to stimulate MMP-2 production and secretion, respectively. Quantitative PCR revealed that this effect was not attributed to transcriptional alterations. This study showed that N-glycosylation was a prerequisite for efficient MMP-2 production, with the IgII loop domain contributing significantly to this process. Perturbation of the function of IgII-EMMPRIN loop could have potential therapeutic value in the inhibition of MMP-2-dependent cancer cell invasion and metastasis.

SIKLODEKSTRIN GLIKOSIL TRANSFERASE DAN PEMANFAATANNYA DALAM INDUSTRI [Cyclodextrin Glycosyl Transferase and its application in industries

Directory of Open Access Journals (Sweden)

Budiasih Wahyuntari

2005-12-01

Full Text Available Cyclodextrin glycosyl transferase (CGT-ase is mainly produced by Bacilli. Systematical name of the enzyme is E.C. 2.4.1.19 a-1,4 glucan-4-glycosyl transferase. The enzyme catalyzes hydrolysis of starch intramolecular, and intermolecular transglycosylation of a-1,4, glucan chains. Cyclodextrins are a-1,4 linked cyclic oligosaccharides resulting from enzymatic degradation of starch by cyclodextrin glycosyl transferase through untramolecular transglycosylation. The major cyclodextrins are made up of 6, 7 and 8 glucopyranose units which are known as a-, b-, and y-cyclodextrin. All CGT-ase catalyze three kinds of cyclodextrins, the proportion of the cyclodextrins depends on the enzyme source and reaction conditions. The intermolecular transglycosylation ability of the enzyme has been applied in transfering glycosyl residues into suitable acceptor. Transglycosylation by the enzymes have been tested to improve solubility of some flavonoids and to favor precipitation ci some glycosides.

Metagenomics for the discovery of novel biosurfactants of environmental interest from marine ecosystems.

Science.gov (United States)

Jackson, Stephen A; Borchert, Erik; O'Gara, Fergal; Dobson, Alan D W

2015-06-01

Research focused on the search for new biosurfactants aims to replace chemical surfactants, which while being cost-effective are ecologically undesirable. Metagenomics can lead to discovery of novel biosurfactants, tackling issues of low production yields. Recent successes include the heterologous production of biosurfactants. The dearth of biosurfactants discovered to date through metagenomics is puzzling given that good screening systems and heterologous host systems are available. Copyright © 2015 Elsevier Ltd. All rights reserved.

Metagenomic analysis of the airborne environment in urban spaces.

Science.gov (United States)

Be, Nicholas A; Thissen, James B; Fofanov, Viacheslav Y; Allen, Jonathan E; Rojas, Mark; Golovko, George; Fofanov, Yuriy; Koshinsky, Heather; Jaing, Crystal J

2015-02-01

The organisms in aerosol microenvironments, especially densely populated urban areas, are relevant to maintenance of public health and detection of potential epidemic or biothreat agents. To examine aerosolized microorganisms in this environment, we performed sequencing on the material from an urban aerosol surveillance program. Whole metagenome sequencing was applied to DNA extracted from air filters obtained during periods from each of the four seasons. The composition of bacteria, plants, fungi, invertebrates, and viruses demonstrated distinct temporal shifts. Bacillus thuringiensis serovar kurstaki was detected in samples known to be exposed to aerosolized spores, illustrating the potential utility of this approach for identification of intentionally introduced microbial agents. Together, these data demonstrate the temporally dependent metagenomic complexity of urban aerosols and the potential of genomic analytical techniques for biosurveillance and monitoring of threats to public health.

Functional Divergence in the Role of N-Linked Glycosylation in Smoothened Signaling.

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Suresh Marada

2015-08-01

Full Text Available The G protein-coupled receptor (GPCR Smoothened (Smo is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice.

Finding the needles in the meta-genome haystack

NARCIS (Netherlands)

Kowalchuk, G.A.; Speksnijder, A.G.C.L.; Zhang, K.; Goodman, R.M.; Veen, van J.A.

2007-01-01

In the collective genomes (the metagenome) of the microorganisms inhabiting the Earth's diverse environments is written the history of life on this planet. New molecular tools developed and used for the past 15 years by microbial ecologists are facilitating the extraction, cloning, screening, and

Por secretion system-dependent secretion and glycosylation of Porphyromonas gingivalis hemin-binding protein 35.

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Mikio Shoji

Full Text Available The anaerobic Gram-negative bacterium Porphyromonas gingivalis is a major pathogen in severe forms of periodontal disease and refractory periapical perodontitis. We have recently found that P. gingivalis has a novel secretion system named the Por secretion system (PorSS, which is responsible for secretion of major extracellular proteinases, Arg-gingipains (Rgps and Lys-gingipain. These proteinases contain conserved C-terminal domains (CTDs in their C-termini. Hemin-binding protein 35 (HBP35, which is one of the outer membrane proteins of P. gingivalis and contributes to its haem utilization, also contains a CTD, suggesting that HBP35 is translocated to the cell surface via the PorSS. In this study, immunoblot analysis of P. gingivalis mutants deficient in the PorSS or in the biosynthesis of anionic polysaccharide-lipopolysaccharide (A-LPS revealed that HBP35 is translocated to the cell surface via the PorSS and is glycosylated with A-LPS. From deletion analysis with a GFP-CTD[HBP35] green fluorescent protein fusion, the C-terminal 22 amino acid residues of CTD[HBP35] were found to be required for cell surface translocation and glycosylation. The GFP-CTD fusion study also revealed that the CTDs of CPG70, peptidylarginine deiminase, P27 and RgpB play roles in PorSS-dependent translocation and glycosylation. However, CTD-region peptides were not found in samples of glycosylated HBP35 protein by peptide map fingerprinting analysis, and antibodies against CTD-regions peptides did not react with glycosylated HBP35 protein. These results suggest both that the CTD region functions as a recognition signal for the PorSS and that glycosylation of CTD proteins occurs after removal of the CTD region. Rabbits were used for making antisera against bacterial proteins in this study.

Moleculo Long-Read Sequencing Facilitates Assembly and Genomic Binning from Complex Soil Metagenomes

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White, Richard Allen; Bottos, Eric M.; Roy Chowdhury, Taniya; Zucker, Jeremy D.; Brislawn, Colin J.; Nicora, Carrie D.; Fansler, Sarah J.; Glaesemann, Kurt R.; Glass, Kevin; Jansson, Janet K.; Langille, Morgan

2016-06-28

Soil metagenomics has been touted as the “grand challenge” for metagenomics, as the high microbial diversity and spatial heterogeneity of soils make them unamenable to current assembly platforms. Here, we aimed to improve soil metagenomic sequence assembly by applying the Moleculo synthetic long-read sequencing technology. In total, we obtained 267 Gbp of raw sequence data from a native prairie soil; these data included 109.7 Gbp of short-read data (~100 bp) from the Joint Genome Institute (JGI), an additional 87.7 Gbp of rapid-mode read data (~250 bp), plus 69.6 Gbp (>1.5 kbp) from Moleculo sequencing. The Moleculo data alone yielded over 5,600 reads of >10 kbp in length, and over 95% of the unassembled reads mapped to contigs of >1.5 kbp. Hybrid assembly of all data resulted in more than 10,000 contigs over 10 kbp in length. We mapped three replicate metatranscriptomes derived from the same parent soil to the Moleculo subassembly and found that 95% of the predicted genes, based on their assignments to Enzyme Commission (EC) numbers, were expressed. The Moleculo subassembly also enabled binning of >100 microbial genome bins. We obtained via direct binning the first complete genome, that of “CandidatusPseudomonas sp. strain JKJ-1” from a native soil metagenome. By mapping metatranscriptome sequence reads back to the bins, we found that several bins corresponding to low-relative-abundanceAcidobacteriawere highly transcriptionally active, whereas bins corresponding to high-relative-abundanceVerrucomicrobiawere not. These results demonstrate that Moleculo sequencing provides a significant advance for resolving complex soil microbial communities.

IMPORTANCESoil microorganisms carry out key processes for life on our planet, including cycling of carbon and other nutrients and supporting growth of plants. However, there is poor molecular-level understanding of their

Forest harvesting reduces the soil metagenomic potential for biomass decomposition.

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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.

Adaptive antibody diversification through N-linked glycosylation of the immunoglobulin variable region.

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van de Bovenkamp, Fleur S; Derksen, Ninotska I L; Ooijevaar-de Heer, Pleuni; van Schie, Karin A; Kruithof, Simone; Berkowska, Magdalena A; van der Schoot, C Ellen; IJspeert, Hanna; van der Burg, Mirjam; Gils, Ann; Hafkenscheid, Lise; Toes, René E M; Rombouts, Yoann; Plomp, Rosina; Wuhrer, Manfred; van Ham, S Marieke; Vidarsson, Gestur; Rispens, Theo

2018-02-20

A hallmark of B-cell immunity is the generation of a diverse repertoire of antibodies from a limited set of germline V(D)J genes. This repertoire is usually defined in terms of amino acid composition. However, variable domains may also acquire N -linked glycans, a process conditional on the introduction of consensus amino acid motifs ( N -glycosylation sites) during somatic hypermutation. High levels of variable domain glycans have been associated with autoantibodies in rheumatoid arthritis, as well as certain follicular lymphomas. However, the role of these glycans in the humoral immune response remains poorly understood. Interestingly, studies have reported both positive and negative effects on antibody affinity. Our aim was to elucidate the role of variable domain glycans during antigen-specific antibody responses. By analyzing B-cell repertoires by next-generation sequencing, we demonstrate that N -glycosylation sites are introduced at positions in which glycans can affect antigen binding as a result of a specific clustering of progenitor glycosylation sites in the germline sequences of variable domain genes. By analyzing multiple human monoclonal and polyclonal (auto)antibody responses, we subsequently show that this process is subject to selection during antigen-specific antibody responses, skewed toward IgG4, and positively contributes to antigen binding. Together, these results highlight a physiological role for variable domain glycosylation as an additional layer of antibody diversification that modulates antigen binding.

Enhancing Accuracy in Molecular Weight Determination of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass Spectrometry

NARCIS (Netherlands)

Wang, Guanbo; de Jong, Rob N; van den Bremer, Ewald T J; Parren, Paul W H I; Heck, Albert J R

2017-01-01

The determination of molecular weights (MWs) of heavily glycosylated proteins is seriously hampered by the physicochemical characteristics and heterogeneity of the attached carbohydrates. Glycosylation impacts protein migration during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis

Role of Cytokine-Induced Glycosylation Changes in Regulating Cell Interactions and Cell Signaling in Inflammatory Diseases and Cancer

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Justine H. Dewald

2016-11-01

Full Text Available Glycosylation is one of the most important modifications of proteins and lipids, and cell surface glycoconjugates are thought to play important roles in a variety of biological functions including cell-cell and cell-substrate interactions, bacterial adhesion, cell immunogenicity and cell signaling. Alterations of glycosylation are observed in number of diseases such as cancer and chronic inflammation. In that context, pro-inflammatory cytokines have been shown to modulate cell surface glycosylation by regulating the expression of glycosyltransferases involved in the biosynthesis of carbohydrate chains. These changes in cell surface glycosylation are also known to regulate cell signaling and could contribute to disease pathogenesis. This review summarizes our current knowledge of the glycosylation changes induced by pro-inflammatory cytokines, with a particular focus on cancer and cystic fibrosis, and their consequences on cell interactions and signaling.

General N-and O-Linked Glycosylation of Lipoproteins in Mycoplasmas and Role of Exogenous Oligosaccharide.

Science.gov (United States)

Daubenspeck, James M; Jordan, David S; Simmons, Warren; Renfrow, Matthew B; Dybvig, Kevin

2015-01-01

The lack of a cell wall, flagella, fimbria, and other extracellular appendages and the possession of only a single membrane render the mycoplasmas structurally simplistic and ideal model organisms for the study of glycoconjugates. Most species have genomes of about 800 kb and code for few proteins predicted to have a role in glycobiology. The murine pathogens Mycoplasma arthritidis and Mycoplasma pulmonis have only a single gene annotated as coding for a glycosyltransferase but synthesize glycolipid, polysaccharide and glycoproteins. Previously, it was shown that M. arthritidis glycosylated surface lipoproteins through O-linkage. In the current study, O-linked glycoproteins were similarly found in M. pulmonis and both species of mycoplasma were found to also possess N-linked glycans at residues of asparagine and glutamine. Protein glycosylation occurred at numerous sites on surface-exposed lipoproteins with no apparent amino acid sequence specificity. The lipoproteins of Mycoplasma pneumoniae also are glycosylated. Glycosylation was dependent on the glycosidic linkages from host oligosaccharides. As far as we are aware, N-linked glycoproteins have not been previously described in Gram-positive bacteria, the organisms to which the mycoplasmas are phylogenetically related. The findings indicate that the mycoplasma cell surface is heavily glycosylated with implications for the modulation of mycoplasma-host interactions.

Enrichment allows identification of diverse, rare elements in metagenomic resistome-virulome sequencing.

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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 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.

SnapShot: O-Glycosylation Pathways across Kingdoms

DEFF Research Database (Denmark)

Joshi, Hiren J.; Narimatsu, Yoshiki; Schjoldager, Katrine T.

2018-01-01

O-glycosylation is one of the most abundant and diverse types of post-translational modifications of proteins. O-glycans modulate the structure, stability, and function of proteins and serve generalized as well as highly specific roles in most biological processes. This ShapShot presents types of......-glycans found in different organisms and their principle biosynthetic pathways...

Role of protein glycosylation on the expression of muscarinic receptors of N4TG1 neuroblastoma cells

International Nuclear Information System (INIS)

Ahmad, A.; Chiang, P.K.

1986-01-01

Muscarinic acetylcholine receptors (mAChR) are glycoproteins. Experiments were conducted to determine whether active glycosylation of proteins in N4TG1 neuroblastoma cells could affect the expression of muscarinic receptors on the cell surface. The binding of radioactive N-methylscopolamine, a membrane impermeable ligand, to intact cells was used as a measure of mAChR. In the presence of the inhibitors of glycosylation, such as tunicamycin, monensin and amphomycin, N-linked glycosylation of proteins in the N4TG1 cells was inhibited, as measured by the incorporation of radioactive glucosamine or mannose in proteins. At the concentrations of tunicamycin and monensin used, the glycosylation of proteins after 3 hours were drastically reduced, but the number of mAChR in the cells was not altered. The apparent lack of effect within a short incubation period could be attributed to the presence of preformed oligosaccharide dolichol readily available for N-glycosylation. However, after 24 hours, tunicamycin (0.05 μg/ml) caused a decrease in the number of mAChR by 17% without having any effect on protein synthesis. Therefore, de novo glycosylation of proteins may be required for the expression of mAChR receptors in the N4TG1 neuroblastoma cell surface

An enzymatic deglycosylation scheme enabling identification of core fucosylated N-glycans and O-glycosylation site mapping of human plasma proteins

DEFF Research Database (Denmark)

Hägglund, Per; Matthiesen, R.; Elortza, F.

2007-01-01

and N-acetyl-β-glucosaminidase) are also included. The two strategies were here applied to identify 103 N-glycosylation sites in the Cohn IV fraction of human plasma. In addition, Endo D/H digestion uniquely enabled identification of 23 fucosylated N-glycosylation sites. Several O-glycosylated peptides......, thereby reducing the complexity and facilitating glycosylation site determinations. Here, we have used two different enzymatic deglycosylation strategies for N-glycosylation site analysis. (1) Removal of entire N-glycan chains by peptide- N-glycosidase (PNGase) digestion, with concomitant deamidation...... of the released asparagine residue. The reaction is carried out in H218O to facilitate identification of the formerly glycosylated peptide by incorporatation of 18O into the formed aspartic acid residue. (2) Digestion with two endo-β- N-acetylglucosaminidases (Endo D and Endo H) that cleave the glycosidic bond...

Glycosylation Alters Dimerization Properties of a Cell-surface Signaling Protein, Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1)*

Science.gov (United States)

Zhuo, You; Yang, Jeong-Yeh; Moremen, Kelley W.; Prestegard, James H.

2016-01-01

Human carcinoembryonic antigen-related cell adhesion molecule 1 (C?/Au: EACAM1) is a cell-surface signaling molecule involved in cell adhesion, proliferation, and immune response. It is also implicated in cancer angiogenesis, progression, and metastasis. This diverse set of effects likely arises as a result of the numerous homophilic and heterophilic interactions that CEACAM1 can have with itself and other molecules. Its N-terminal Ig variable (IgV) domain has been suggested to be a principal player in these interactions. Previous crystal structures of the β-sandwich-like IgV domain have been produced using Escherichia coli-expressed material, which lacks native glycosylation. These have led to distinctly different proposals for dimer interfaces, one involving interactions of ABED β-strands and the other involving GFCC′C″ β-strands, with the former burying one prominent glycosylation site. These structures raise questions as to which form may exist in solution and what the effect of glycosylation may have on this form. Here, we use NMR cross-correlation measurements to examine the effect of glycosylation on CEACAM1-IgV dimerization and use residual dipolar coupling (RDC) measurements to characterize the solution structure of the non-glycosylated form. Our findings demonstrate that even addition of a single N-linked GlcNAc at potential glycosylation sites inhibits dimer formation. Surprisingly, RDC data collected on E. coli expressed material in solution indicate that a dimer using the non-glycosylated GFCC′C″ interface is preferred even in the absence of glycosylation. The results open new questions about what other factors may facilitate dimerization of CEACAM1 in vivo, and what roles glycosylation may play in heterophylic interactions. PMID:27471271

Modulation and modeling of monoclonal antibody N-linked glycosylation in mammalian cell perfusion reactors.

Science.gov (United States)

Karst, Daniel J; Scibona, Ernesto; Serra, Elisa; Bielser, Jean-Marc; Souquet, Jonathan; Stettler, Matthieu; Broly, Hervé; Soos, Miroslav; Morbidelli, Massimo; Villiger, Thomas K

2017-09-01

Mammalian cell perfusion cultures are gaining renewed interest as an alternative to traditional fed-batch processes for the production of therapeutic proteins, such as monoclonal antibodies (mAb). The steady state operation at high viable cell density allows the continuous delivery of antibody product with increased space-time yield and reduced in-process variability of critical product quality attributes (CQA). In particular, the production of a confined mAb N-linked glycosylation pattern has the potential to increase therapeutic efficacy and bioactivity. In this study, we show that accurate control of flow rates, media composition and cell density of a Chinese hamster ovary (CHO) cell perfusion bioreactor allowed the production of a constant glycosylation profile for over 20 days. Steady state was reached after an initial transition phase of 6 days required for the stabilization of extra- and intracellular processes. The possibility to modulate the glycosylation profile was further investigated in a Design of Experiment (DoE), at different viable cell density and media supplement concentrations. This strategy was implemented in a sequential screening approach, where various steady states were achieved sequentially during one culture. It was found that, whereas high ammonia levels reached at high viable cell densities (VCD) values inhibited the processing to complex glycan structures, the supplementation of either galactose, or manganese as well as their synergy significantly increased the proportion of complex forms. The obtained experimental data set was used to compare the reliability of a statistical response surface model (RSM) to a mechanistic model of N-linked glycosylation. The latter outperformed the response surface predictions with respect to its capability and reliability in predicting the system behavior (i.e., glycosylation pattern) outside the experimental space covered by the DoE design used for the model parameter estimation. Therefore, we can

Enhanced SCAP glycosylation by inflammation induces macrophage foam cell formation.

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Chao Zhou

Full Text Available Inflammatory stress promotes foam cell formation by disrupting LDL receptor feedback regulation in macrophages. Sterol Regulatory Element Binding Proteins (SREBPs Cleavage-Activating Protein (SCAP glycosylation plays crucial roles in regulating LDL receptor and 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCoAR feedback regulation. The present study was to investigate if inflammatory stress disrupts LDL receptor and HMGCoAR feedback regulation by affecting SCAP glycosylation in THP-1 macrophages. Intracellular cholesterol content was assessed by Oil Red O staining and quantitative assay. The expression of molecules controlling cholesterol homeostasis was examined using real-time quantitative RT-PCR and Western blotting. The translocation of SCAP from the endoplasmic reticulum (ER to the Golgi was detected by confocal microscopy. We demonstrated that exposure to inflammatory cytokines increased lipid accumulation in THP-1 macrophages, accompanying with an increased SCAP expression even in the presence of a high concentration of LDL. These inflammatory cytokines also prolonged the half-life of SCAP by enhancing glycosylation of SCAP due to the elevated expression of the Golgi mannosidase II. This may enhance translocation and recycling of SCAP between the ER and the Golgi, escorting more SREBP2 from the ER to the Golgi for activation by proteolytic cleavages as evidenced by an increased N-terminal of SREBP2 (active form. As a consequence, the LDL receptor and HMGCoAR expression were up-regulated. Interestingly, these effects could be blocked by inhibitors of Golgi mannosidases. Our results indicated that inflammation increased native LDL uptake and endogenous cholesterol de novo synthesis, thereby causing foam cell formation via increasing transcription and protein glycosylation of SCAP in macrophages. These data imply that inhibitors of Golgi processing enzymes might have a potential vascular-protective role in prevention of atherosclerotic foam

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

Science.gov (United States)

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.

Stannylene‐Mediated Regioselective 6‐O‐Glycosylation of Unprotected Phenyl 1‐Thioglycopyranosides

DEFF Research Database (Denmark)

Maggi, Agnese; Madsen, Robert

2013-01-01

acetal, and then subjected to selective glycosylation at the 6‐position with the Koenigs–Knorr protocol. Peracylated glycosyl bromides of D‐glucose, D‐galactose, D‐mannose and D‐glucosamine were employed as the donors to give the corresponding (1→6)‐linked disaccharides in moderate to good yields......‐thio‐β‐D‐glucopyranoside gave rise to the corresponding (1→6)‐linked trisaccharides in moderate yields....

GtfA and GtfB Are Both Required for Protein O-Glycosylation in Lactobacillus plantarum

Science.gov (United States)

Lee, I-Chiao; van Swam, Iris I.; Tomita, Satoru; Morsomme, Pierre; Rolain, Thomas; Hols, Pascal; Bron, Peter A.

2014-01-01

Acm2, the major autolysin of Lactobacillus plantarum WCFS1, was recently found to be O-glycosylated with N-acetylhexosamine, likely N-acetylglucosamine (GlcNAc). In this study, we set out to identify the glycosylation machinery by employing a comparative genomics approach to identify Gtf1 homologues, which are involved in fimbria-associated protein 1 (Fap1) glycosylation in Streptococcus parasanguinis. This in silico approach resulted in the identification of 6 candidate L. plantarum WCFS1 genes with significant homology to Gtf1, namely, tagE1 to tagE6. These candidate genes were targeted by systematic gene deletion, followed by assessment of the consequences on glycosylation of Acm2. We observed a changed mobility of Acm2 on SDS-PAGE in the tagE5E6 deletion strain, while deletion of other tagE genes resulted in Acm2 mobility comparable to that of the wild type. Subsequent mass spectrometry analysis of excised and in-gel-digested Acm2 confirmed the loss of glycosylation on Acm2 in the tagE5E6 deletion mutant, whereas a lectin blot using GlcNAc-specific succinylated wheat germ agglutinin (sWGA) revealed that besides Acm2, tagE5E6 deletion also abolished all but one other sWGA-reactive, protease-sensitive signal. Only complementation of both tagE5 and tagE6 restored those sWGA lectin signals, establishing that TagE5 and TagE6 are both required for the glycosylation of Acm2 as well as the vast majority of other sWGA-reactive proteins. Finally, sWGA lectin blotting experiments using a panel of 8 other L. plantarum strains revealed that protein glycosylation is a common feature in L. plantarum strains. With the establishment of these enzymes as protein glycosyltransferases, we propose to rename TagE5 and TagE6 as GtfA and GtfB, respectively. PMID:24532775

Transgenic rice seed expressing flavonoid biosynthetic genes accumulate glycosylated and/or acylated flavonoids in protein bodies

Science.gov (United States)

Ogo, Yuko; Mori, Tetsuya; Nakabayashi, Ryo; Saito, Kazuki; Takaiwa, Fumio

2016-01-01

Plant-specialized (or secondary) metabolites represent an important source of high-value chemicals. In order to generate a new production platform for these metabolites, an attempt was made to produce flavonoids in rice seeds. Metabolome analysis of these transgenic rice seeds using liquid chromatography-photodiode array-quadrupole time-of-flight mass spectrometry was performed. A total of 4392 peaks were detected in both transgenic and non-transgenic rice, 20–40% of which were only detected in transgenic rice. Among these, 82 flavonoids, including 37 flavonols, 11 isoflavones, and 34 flavones, were chemically assigned. Most of the flavonols and isoflavones were O-glycosylated, while many flavones were O-glycosylated and/or C-glycosylated. Several flavonoids were acylated with malonyl, feruloyl, acetyl, and coumaroyl groups. These glycosylated/acylated flavonoids are thought to have been biosynthesized by endogenous rice enzymes using newly synthesized flavonoids whose biosynthesis was catalysed by exogenous enzymes. The subcellular localization of the flavonoids differed depending on the class of aglycone and the glycosylation/acylation pattern. Therefore, flavonoids with the intended aglycones were efficiently produced in rice seeds via the exogenous enzymes introduced, while the flavonoids were variously glycosylated/acylated by endogenous enzymes. The results suggest that rice seeds are useful not only as a production platform for plant-specialized metabolites such as flavonoids but also as a tool for expanding the diversity of flavonoid structures, providing novel, physiologically active substances. PMID:26438413

Discrimination between glycosylation patterns of therapeutic antibodies using a microfluidic platform, MALDI-MS and multivariate statistics.

Science.gov (United States)

Thuy, Tran Thi; Tengstrand, Erik; Aberg, Magnus; Thorsén, Gunnar

2012-11-01

Optimal glycosylation with respect to the efficacy, serum half-life time, and immunogenic properties is essential in the generation of therapeutic antibodies. The glycosylation pattern can be affected by several different parameters during the manufacture of antibodies and may change significantly over cultivation time. Fast and robust methods for determination of the glycosylation patterns of therapeutic antibodies are therefore needed. We have recently presented an efficient method for the determination of glycans on therapeutic antibodies using a microfluidic CD platform for sample preparation prior to matrix-assisted laser-desorption mass spectrometry analysis. In the present work, this method is applied to analyse the glycosylation patterns of three commercially available therapeutic antibodies and one intended for therapeutic use. Two of the antibodies produced in mouse myeloma cell line (SP2/0) and one produced in Chinese hamster ovary (CHO) cells exhibited similar glycosylation patterns but could still be readily differentiated from each other using multivariate statistical methods. The two antibodies with most similar glycosylation patterns were also studied in an assessment of the method's applicability for quality control of therapeutic antibodies. The method presented in this paper is highly automated and rapid. It can therefore efficiently generate data that helps to keep a production process within the desired design space or assess that an identical product is being produced after changes to the process. Copyright © 2012 Elsevier B.V. All rights reserved.

Genomic and metagenomic challenges and opportunities for bioleaching: a mini-review.

Science.gov (United States)

Cárdenas, Juan Pablo; Quatrini, Raquel; Holmes, David S

2016-09-01

High-throughput genomic technologies are accelerating progress in understanding the diversity of microbial life in many environments. Here we highlight advances in genomics and metagenomics of microorganisms from bioleaching heaps and related acidic mining environments. Bioleaching heaps used for copper recovery provide significant opportunities to study the processes and mechanisms underlying microbial successions and the influence of community composition on ecosystem functioning. Obtaining quantitative and process-level knowledge of these dynamics is pivotal for understanding how microorganisms contribute to the solubilization of copper for industrial recovery. Advances in DNA sequencing technology provide unprecedented opportunities to obtain information about the genomes of bioleaching microorganisms, allowing predictive models of metabolic potential and ecosystem-level interactions to be constructed. These approaches are enabling predictive phenotyping of organisms many of which are recalcitrant to genetic approaches or are unculturable. This mini-review describes current bioleaching genomic and metagenomic projects and addresses the use of genome information to: (i) build metabolic models; (ii) predict microbial interactions; (iii) estimate genetic diversity; and (iv) study microbial evolution. Key challenges and perspectives of bioleaching genomics/metagenomics are addressed. Copyright © 2016 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.

Glycosylation of Hemagglutinin and Neuraminidase of Influenza A Virus as Signature for Ecological Spillover and Adaptation among Influenza Reservoirs

Directory of Open Access Journals (Sweden)

Paul Kim

2018-04-01

Full Text Available Glycosylation of the hemagglutinin (HA and neuraminidase (NA of the influenza provides crucial means for immune evasion and viral fitness in a host population. However, the time-dependent dynamics of each glycosylation sites have not been addressed. We monitored the potential N-linked glycosylation (NLG sites of over 10,000 HA and NA of H1N1 subtype isolated from human, avian, and swine species over the past century. The results show a shift in glycosylation sites as a hallmark of 1918 and 2009 pandemics, and also for the 1976 “abortive pandemic”. Co-segregation of particular glycosylation sites was identified as a characteristic of zoonotic transmission from animal reservoirs, and interestingly, of “reverse zoonosis” of human viruses into swine populations as well. After the 2009 pandemic, recent isolates accrued glycosylation at canonical sites in HA, reflecting gradual seasonal adaptation, and a novel glycosylation in NA as an independent signature for adaptation among humans. Structural predictions indicated a remarkably pleiotropic influence of glycans on multiple HA epitopes for immune evasion, without sacrificing the receptor binding of HA or the activity of NA. The results provided the rationale for establishing the ecological niche of influenza viruses among the reservoir and could be implemented for influenza surveillance and improving pandemic preparedness.

Glycosylation of Hemagglutinin and Neuraminidase of Influenza A Virus as Signature for Ecological Spillover and Adaptation among Influenza Reservoirs

Science.gov (United States)

Kim, Paul; Jang, Yo Han; Kwon, Soon Bin; Lee, Chung Min; Han, Gyoonhee; Seong, Baik Lin

2018-01-01

Glycosylation of the hemagglutinin (HA) and neuraminidase (NA) of the influenza provides crucial means for immune evasion and viral fitness in a host population. However, the time-dependent dynamics of each glycosylation sites have not been addressed. We monitored the potential N-linked glycosylation (NLG) sites of over 10,000 HA and NA of H1N1 subtype isolated from human, avian, and swine species over the past century. The results show a shift in glycosylation sites as a hallmark of 1918 and 2009 pandemics, and also for the 1976 “abortive pandemic”. Co-segregation of particular glycosylation sites was identified as a characteristic of zoonotic transmission from animal reservoirs, and interestingly, of “reverse zoonosis” of human viruses into swine populations as well. After the 2009 pandemic, recent isolates accrued glycosylation at canonical sites in HA, reflecting gradual seasonal adaptation, and a novel glycosylation in NA as an independent signature for adaptation among humans. Structural predictions indicated a remarkably pleiotropic influence of glycans on multiple HA epitopes for immune evasion, without sacrificing the receptor binding of HA or the activity of NA. The results provided the rationale for establishing the ecological niche of influenza viruses among the reservoir and could be implemented for influenza surveillance and improving pandemic preparedness. PMID:29642453

The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants

DEFF Research Database (Denmark)

Behrendt, N; Rønne, E; Ploug, M

1990-01-01

-PA. The purified protein shows a single 55-60 kDa band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. It is a heavily glycosylated protein, the deglycosylated polypeptide chain comprising only 35 kDa. The glycosylated protein contains N-acetyl-D-glucosamine and sialic acid......, but no N-acetyl-D-galactosamine. Glycosylation is responsible for substantial heterogeneity in the receptor on phorbol ester-stimulated U937 cells, and also for molecular weight variations among various cell lines. The amino acid composition and the NH2-terminal amino acid sequence are reported...

Sensitive and comprehensive analysis of O-glycosylation in biotherapeutics: a case study of novel erythropoiesis stimulating protein.

Science.gov (United States)

Kim, Unyong; Oh, Myung Jin; Seo, Youngsuk; Jeon, Yinae; Eom, Joon-Ho; An, Hyun Joo

2017-09-01

Glycosylation of recombinant human erythropoietins (rhEPOs) is significantly associated with drug's quality and potency. Thus, comprehensive characterization of glycosylation is vital to assess the biotherapeutic quality and establish the equivalency of biosimilar rhEPOs. However, current glycan analysis mainly focuses on the N-glycans due to the absence of analytical tools to liberate O-glycans with high sensitivity. We developed selective and sensitive method to profile native O-glycans on rhEPOs. O-glycosylation on rhEPO including O-acetylation on a sialic acid was comprehensively characterized. Details such as O-glycan structure and O-acetyl-modification site were obtained from tandem MS. This method may be applied to QC and batch analysis of not only rhEPOs but also other biotherapeutics bearing multiple O-glycosylations.

Toward a Standards-Compliant Genomic and Metagenomic Publication Record

DEFF Research Database (Denmark)

Garrity, GM; Field, D; Kyrpides, N

2008-01-01

Increasingly, we are aware as a community of the growing need to manage the avalanche of genomic and metagenomic data, in addition to related data types like ribosomal RNA and barcode sequences, in a way that tightly integrates contextual data with traditional literature in a machine-readable way...... is in the midst of a publishing revolution. This revolution is marked by a growing shift away from a traditional dichotomy between "journal articles" and "database entries" and an increasing adoption of hybrid models of collecting and disseminating scientific information. With respect to genomes and metagenomes...... or communities) such as the call by the GSC for a central repository of Standard Operating Procedures describing the genomic annotation pipelines of the major sequencing centers. We argue that such an "eJournal," published under the Open Access paradigm by the GSC, could be an attractive publishing forum...

Mining the Virgin Land of Neurotoxicology: A Novel Paradigm of Neurotoxic Peptides Action on Glycosylated Voltage-Gated Sodium Channels

Directory of Open Access Journals (Sweden)

Zhirui Liu

2012-01-01

Full Text Available Voltage-gated sodium channels (VGSCs are important membrane protein carrying on the molecular basis for action potentials (AP in neuronal firings. Even though the structure-function studies were the most pursued spots, the posttranslation modification processes, such as glycosylation, phosphorylation, and alternative splicing associating with channel functions captured less eyesights. The accumulative research suggested an interaction between the sialic acids chains and ion-permeable pores, giving rise to subtle but significant impacts on channel gating. Sodium channel-specific neurotoxic toxins, a family of long-chain polypeptides originated from venomous animals, are found to potentially share the binding sites adjacent to glycosylated region on VGSCs. Thus, an interaction between toxin and glycosylated VGSC might hopefully join the campaign to approach the role of glycosylation in modulating VGSCs-involved neuronal network activity. This paper will cover the state-of-the-art advances of researches on glycosylation-mediated VGSCs function and the possible underlying mechanisms of interactions between toxin and glycosylated VGSCs, which may therefore, fulfill the knowledge in identifying the pharmacological targets and therapeutic values of VGSCs.

Evaluation of FTA ® paper for storage of oral meta-genomic DNA.

Science.gov (United States)

Foitzik, Magdalena; Stumpp, Sascha N; Grischke, Jasmin; Eberhard, Jörg; Stiesch, Meike

2014-10-01

The purpose of the present study was to evaluate the short-term storage of meta-genomic DNA from native oral biofilms on FTA(®) paper. Thirteen volunteers of both sexes received an acrylic splint for intraoral biofilm formation over a period of 48 hours. The biofilms were collected, resuspended in phosphate-buffered saline, and either stored on FTA(®) paper or directly processed by standard laboratory DNA extraction. The nucleic acid extraction efficiencies were evaluated by 16S rDNA targeted SSCP fingerprinting. The acquired banding pattern of FTA-derived meta-genomic DNA was compared to a standard DNA preparation protocol. Sensitivity and positive predictive values were calculated. The volunteers showed inter-individual differences in their bacterial species composition. A total of 200 bands were found for both methods and 85% of the banding patterns were equal, representing a sensitivity of 0.941 and a false-negative predictive value of 0.059. Meta-genomic DNA sampling, extraction, and adhesion using FTA(®) paper is a reliable method for storage of microbial DNA for a short period of time.

Separating metagenomic short reads into genomes via clustering

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Tanaseichuk Olga

2012-09-01

Full Text Available Abstract Background The metagenomics approach allows the simultaneous sequencing of all genomes in an environmental sample. This results in high complexity datasets, where in addition to repeats and sequencing errors, the number of genomes and their abundance ratios are unknown. Recently developed next-generation sequencing (NGS technologies significantly improve the sequencing efficiency and cost. On the other hand, they result in shorter reads, which makes the separation of reads from different species harder. Among the existing computational tools for metagenomic analysis, there are similarity-based methods that use reference databases to align reads and composition-based methods that use composition patterns (i.e., frequencies of short words or l-mers to cluster reads. Similarity-based methods are unable to classify reads from unknown species without close references (which constitute the majority of reads. Since composition patterns are preserved only in significantly large fragments, composition-based tools cannot be used for very short reads, which becomes a significant limitation with the development of NGS. A recently proposed algorithm, AbundanceBin, introduced another method that bins reads based on predicted abundances of the genomes sequenced. However, it does not separate reads from genomes of similar abundance levels. Results In this work, we present a two-phase heuristic algorithm for separating short paired-end reads from different genomes in a metagenomic dataset. We use the observation that most of the l-mers belong to unique genomes when l is sufficiently large. The first phase of the algorithm results in clusters of l-mers each of which belongs to one genome. During the second phase, clusters are merged based on l-mer repeat information. These final clusters are used to assign reads. The algorithm could handle very short reads and sequencing errors. It is initially designed for genomes with similar abundance levels and then

A feruloyl esterase derived from a leachate metagenome library

CSIR Research Space (South Africa)

Rashamuse, K

2012-01-01

Full Text Available A feruloyl esterase encoding gene (designated fae6), derived from a leachate metagenomic library, was cloned and the nucleotide sequence of the insert DNA determined. Translational analysis revealed that fae6 consists of a 515 amino acid polypeptide...

A probabilistic model to recover individual genomes from metagenomes

NARCIS (Netherlands)

J. Dröge (Johannes); A. Schönhuth (Alexander); A.C. McHardy (Alice)

2017-01-01

textabstractShotgun metagenomics of microbial communities reveal information about strains of relevance for applications in medicine, biotechnology and ecology. Recovering their genomes is a crucial but very challenging step due to the complexity of the underlying biological system and technical

SLC39A8 Deficiency: A Disorder of Manganese Transport and Glycosylation.

Science.gov (United States)

Park, Julien H; Hogrebe, Max; Grüneberg, Marianne; DuChesne, Ingrid; von der Heiden, Ava L; Reunert, Janine; Schlingmann, Karl P; Boycott, Kym M; Beaulieu, Chandree L; Mhanni, Aziz A; Innes, A Micheil; Hörtnagel, Konstanze; Biskup, Saskia; Gleixner, Eva M; Kurlemann, Gerhard; Fiedler, Barbara; Omran, Heymut; Rutsch, Frank; Wada, Yoshinao; Tsiakas, Konstantinos; Santer, René; Nebert, Daniel W; Rust, Stephan; Marquardt, Thorsten

2015-12-03

SLC39A8 is a membrane transporter responsible for manganese uptake into the cell. Via whole-exome sequencing, we studied a child that presented with cranial asymmetry, severe infantile spasms with hypsarrhythmia, and dysproportionate dwarfism. Analysis of transferrin glycosylation revealed severe dysglycosylation corresponding to a type II congenital disorder of glycosylation (CDG) and the blood manganese levels were below the detection limit. The variants c.112G>C (p.Gly38Arg) and c.1019T>A (p.Ile340Asn) were identified in SLC39A8. A second individual with the variants c.97G>A (p.Val33Met) and c.1004G>C (p.Ser335Thr) on the paternal allele and c.610G>T (p.Gly204Cys) on the maternal allele was identified among a group of unresolved case subjects with CDG. These data demonstrate that variants in SLC39A8 impair the function of manganese-dependent enzymes, most notably β-1,4-galactosyltransferase, a Golgi enzyme essential for biosynthesis of the carbohydrate part of glycoproteins. Impaired galactosylation leads to a severe disorder with deformed skull, severe seizures, short limbs, profound psychomotor retardation, and hearing loss. Oral galactose supplementation is a treatment option and results in complete normalization of glycosylation. SLC39A8 deficiency links a trace element deficiency with inherited glycosylation disorders. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

An algorithm for detecting eukaryotic sequences in metagenomic ...

Indian Academy of Sciences (India)

species but also from accidental contamination from the genome of eukaryotic host cells. The latter scenario generally occurs in the case of host-associated metagenomes, e.g. microbes living in human gut. In such cases, one needs to identify and remove contaminating host DNA sequences, since the latter sequences will ...

Synthesis of Curcumin Glycosides with Enhanced Anticancer Properties Using One-Pot Multienzyme Glycosylation Technique.

Science.gov (United States)

Gurung, Rit Bahadur; Gong, So Youn; Dhakal, Dipesh; Le, Tuoi Thi; Jung, Na Rae; Jung, Hye Jin; Oh, Tae Jin; Sohng, Jae Kyung

2017-09-28

Curcumin is a natural polyphenolic compound, widely acclaimed for its antioxidant, antiinflammatory, antibacterial, and anticancerous properties. However, its use has been limited due to its low-aqueous solubility and poor bioavailability, rapid clearance, and low cellular uptake. In order to assess the effect of glycosylation on the pharmacological properties of curcumin, one-pot multienzyme (OPME) chemoenzymatic glycosylation reactions with UDP- α-D-glucose or UDP-α-D-2-deoxyglucose as donor substrate were employed. The result indicated significant conversion of curcumin to its glycosylated derivatives: curcumin 4'- O -β- glucoside, curcumin 4',4''-di- O -β-glucoside, curcumin 4'- O -β-2-deoxyglucoside, and curcumin 4',4''-di- O -β-2-deoxyglucoside. The products were characterized by ultra-fast performance liquid chromatography, high-resolution quadruple-time-of-flight electrospray ionization-mass spectrometry, and NMR analyses. All the products showed improved water solubility and comparable antibacterial activities. Additionally, the curcumin 4'- O -β-glucoside and curcumin 4'- O -β-2-deoxyglucoside showed enhanced anticancer activities compared with the parent aglycone and diglycoside derivatives. This result indicates that glycosylation can be an effective approach for enhancing the pharmaceutical properties of different natural products, such as curcumin.

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

Science.gov (United States)

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.

Glycosyl-Nucleolipids as new bioinspired amphiphiles.

Science.gov (United States)

Latxague, Laurent; Patwa, Amit; Amigues, Eric; Barthélémy, Philippe

2013-09-30

Four new Glycosyl-NucleoLipid (GNL) analogs featuring either a single fluorocarbon or double hydrocarbon chains were synthesized in good yields from azido thymidine as starting material. Physicochemical studies (surface tension measurements, differential scanning calorimetry) indicate that hydroxybutanamide-based GNLs feature endothermic phase transition temperatures like the previously reported double chain glycerol-based GNLs. The second generation of GNFs featuring a free nucleobase reported here presents a better surface activity (lower glim) compared to the first generation of GNFs.

Protein glycosylation in cancers and its potential therapeutic applications in neuroblastoma

Directory of Open Access Journals (Sweden)

Wan-Ling Ho

2016-09-01

Full Text Available Abstract Glycosylation is the most complex post-translational modification of proteins. Altered glycans on the tumor- and host-cell surface and in the tumor microenvironment have been identified to mediate critical events in cancer pathogenesis and progression. Tumor-associated glycan changes comprise increased branching of N-glycans, higher density of O-glycans, generation of truncated versions of normal counterparts, and generation of unusual forms of terminal structures arising from sialylation and fucosylation. The functional role of tumor-associated glycans (Tn, sTn, T, and sLea/x is dependent on the interaction with lectins. Lectins are expressed on the surface of immune cells and endothelial cells or exist as extracellular matrix proteins and soluble adhesion molecules. Expression of tumor-associated glycans is involved in the dysregulation of glycogenes, which mainly comprise glycosyltransferases and glycosidases. Furthermore, genetic and epigenetic mechanisms on many glycogenes are associated with malignant transformation. With better understanding of all aspects of cancer-cell glycomics, many tumor-associated glycans have been utilized for diagnostic, prognostic, and therapeutic purposes. Glycan-based therapeutics has been applied to cancers from breast, lung, gastrointestinal system, melanomas, and lymphomas but rarely to neuroblastomas (NBs. The success of anti-disialoganglioside (GD2, a glycolipid antigen antibodies sheds light on glycan-based therapies for NB and also suggests the possibility of protein glycosylation-based therapies for NB. This review summarizes our understanding of cancer glycobiology with a focus of how protein glycosylation and associated glycosyltransferases affect cellular behaviors and treatment outcome of various cancers, especially NB. Finally, we highlight potential applications of glycosylation in drug and cancer vaccine development for NB.

Applying functional metagenomics to search for novel lignocellulosic enzymes in a microbial consortium derived from a thermophilic composting phase of sugarcane bagasse and cow manure.

Science.gov (United States)

Colombo, Lívia Tavares; de Oliveira, Marcelo Nagem Valério; Carneiro, Deisy Guimarães; de Souza, Robson Assis; Alvim, Mariana Caroline Tocantins; Dos Santos, Josenilda Carlos; da Silva, Cynthia Canêdo; Vidigal, Pedro Marcus Pereira; da Silveira, Wendel Batista; Passos, Flávia Maria Lopes

2016-09-01

Environments where lignocellulosic biomass is naturally decomposed are sources for discovery of new hydrolytic enzymes that can reduce the high cost of enzymatic cocktails for second-generation ethanol production. Metagenomic analysis was applied to discover genes coding carbohydrate-depleting enzymes from a microbial laboratory subculture using a mix of sugarcane bagasse and cow manure in the thermophilic composting phase. From a fosmid library, 182 clones had the ability to hydrolyse carbohydrate. Sequencing of 30 fosmids resulted in 12 contigs encoding 34 putative carbohydrate-active enzymes belonging to 17 glycosyl hydrolase (GH) families. One third of the putative proteins belong to the GH3 family, which includes β-glucosidase enzymes known to be important in the cellulose-deconstruction process but present with low activity in commercial enzyme preparations. Phylogenetic analysis of the amino acid sequences of seven selected proteins, including three β-glucosidases, showed low relatedness with protein sequences deposited in databases. These findings highlight microbial consortia obtained from a mixture of decomposing biomass residues, such as sugar cane bagasse and cow manure, as a rich resource of novel enzymes potentially useful in biotechnology for saccharification of lignocellulosic substrate.

Vinasse fertirrigation alters soil resistome dynamics: an analysis based on metagenomic profiles.

Science.gov (United States)

Braga, Lucas P P; Alves, Rafael F; Dellias, Marina T F; Navarrete, Acacio A; Basso, Thiago O; Tsai, Siu M

2017-01-01

Every year around 300 Gl of vinasse, a by-product of ethanol distillation in sugarcane mills, are flushed into more than 9 Mha of sugarcane cropland in Brazil. This practice links fermentation waste management to fertilization for plant biomass production, and it is known as fertirrigation. Here we evaluate public datasets of soil metagenomes mining for changes in antibiotic resistance genes (ARGs) of soils from sugarcane mesocosms repeatedly amended with vinasse. The metagenomes were annotated using the ResFam database. We found that the abundance of open read frames (ORFs) annotated as ARGs changed significantly across 43 different families ( p -value resistome.

Genes Involved in the Endoplasmic Reticulum N-Glycosylation Pathway of the Red Microalga Porphyridium sp.: A Bioinformatic Study

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Oshrat Levy-Ontman

2014-02-01

Full Text Available N-glycosylation is one of the most important post-translational modifications that influence protein polymorphism, including protein structures and their functions. Although this important biological process has been extensively studied in mammals, only limited knowledge exists regarding glycosylation in algae. The current research is focused on the red microalga Porphyridium sp., which is a potentially valuable source for various applications, such as skin therapy, food, and pharmaceuticals. The enzymes involved in the biosynthesis and processing of N-glycans remain undefined in this species, and the mechanism(s of their genetic regulation is completely unknown. In this study, we describe our pioneering attempt to understand the endoplasmic reticulum N-Glycosylation pathway in Porphyridium sp., using a bioinformatic approach. Homology searches, based on sequence similarities with genes encoding proteins involved in the ER N-glycosylation pathway (including their conserved parts were conducted using the TBLASTN function on the algae DNA scaffold contigs database. This approach led to the identification of 24 encoded-genes implicated with the ER N-glycosylation pathway in Porphyridium sp. Homologs were found for almost all known N-glycosylation protein sequences in the ER pathway of Porphyridium sp.; thus, suggesting that the ER-pathway is conserved; as it is in other organisms (animals, plants, yeasts, etc..

A function-based screen for seeking RubisCO active clones from metagenomes: novel enzymes influencing RubisCO activity.

Science.gov (United States)

Böhnke, Stefanie; Perner, Mirjam

2015-03-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a key enzyme of the Calvin cycle, which is responsible for most of Earth's primary production. Although research on RubisCO genes and enzymes in plants, cyanobacteria and bacteria has been ongoing for years, still little is understood about its regulation and activation in bacteria. Even more so, hardly any information exists about the function of metagenomic RubisCOs and the role of the enzymes encoded on the flanking DNA owing to the lack of available function-based screens for seeking active RubisCOs from the environment. Here we present the first solely activity-based approach for identifying RubisCO active fosmid clones from a metagenomic library. We constructed a metagenomic library from hydrothermal vent fluids and screened 1056 fosmid clones. Twelve clones exhibited RubisCO activity and the metagenomic fragments resembled genes from Thiomicrospira crunogena. One of these clones was further analyzed. It contained a 35.2 kb metagenomic insert carrying the RubisCO gene cluster and flanking DNA regions. Knockouts of twelve genes and two intergenic regions on this metagenomic fragment demonstrated that the RubisCO activity was significantly impaired and was attributed to deletions in genes encoding putative transcriptional regulators and those believed to be vital for RubisCO activation. Our new technique revealed a novel link between a poorly characterized gene and RubisCO activity. This screen opens the door to directly investigating RubisCO genes and respective enzymes from environmental samples.

Blood pressure reduction due to hemoglobin glycosylation in type 2 diabetic patients

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Pedro Cabrales

2008-08-01

Full Text Available Pedro Cabrales1, Miguel A Salazar Vázquez2,3, Beatriz Y Salazar Vázquez3,4, Martha Rodríguez-Morán5, Marcos Intaglietta4, Fernando Guerrero-Romero51La Jolla Bioengineering Institute, La Jolla, California, USA; 2Hospital Regional No. 1, of the Mexican Social Security Institute, Victoria de Durango, Dgo. Mexico; 3Faculty of Medicine and Dept. of Physical Chemistry, Universidad Juárez del Estado de Durango, Victoria de Durango, Dgo. Mexico; 4Department of Bioengineering, University of California, San Diego, La Jolla, California, USA; 5Biomedical Research Unit, of the Mexican Social Security Institute, Victoria de Durango, Dgo. MexicoObjective: To test the hypothesis that glycosylation of hemoglobin constitutes a risk factor for hypertension.Methods: A total of 129 relative uniform diabetic subjects (86 women and 42 men were enrolled in a cross-sectional study. Exclusion criteria included alcohol consumption, smoking, ischemic heart disease, stroke, neoplasia, renal, hepatic, and chronic inflammatory disease. Systolic and diastolic pressures were recorded in subsequent days and mean arterial blood pressure (MAP was determined. Hemoglobin glycosylation was measured by determining the percentage glycosylated hemoglobin (HbA1c by means of the automated microparticle enzyme immunoassay test.Results: MAP was found to be independent of the concentration of HbA1c; however, correcting MAP for the variability in hematocrit, to evidence the level of vasoconstriction (or vasodilatation showed that MAP is negatively correlated with the concentration of HbA1c (p for trend <0.05, when patients treated for hypertension are excluded from the analysis. Patients treated for hypertension showed the opposite trend with increasing MAP as HbA1c increased (p for the difference in trends <0.05.Conclusions: Glycosylation per se appears to lead to blood pressure reduction in type 2 diabetic patients untreated for hypertension. Treatment for hypertension may be

N-Glycosylation of cholera toxin B subunit: serendipity for novel plant-made vaccines?

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Nobuyuki eMatoba

2015-12-01

Full Text Available The non-toxic B subunit of cholera toxin (CTB has attracted considerable interests from vaccinologists due to strong mucosal immunomodulatory effects and potential utility as a vaccine scaffold for heterologous antigens. Along with other conventional protein expression systems, various plant species have been used as recombinant production hosts for CTB and its fusion proteins. However, it has recently become clear that the protein is N-glycosylated within the endoplasmic reticulum of plant cells – a eukaryotic post-translational modification that is not present in native CTB. While functionally active aglycosylated variants have been successfully engineered to circumvent potential safety and regulatory issues related to glycosylation, this modification may actually provide advantageous characteristics to the protein as a vaccine platform. Based on data from our recent studies, I discuss the unique features of N-glycosylated CTB produced in plants for the development of novel vaccines.

Prediction of mucin-type O-glycosylation sites in mammalian proteins using the composition of k-spaced amino acid pairs

Directory of Open Access Journals (Sweden)

Sheng Zhi-Ya

2008-02-01

Full Text Available Abstract Background As one of the most common protein post-translational modifications, glycosylation is involved in a variety of important biological processes. Computational identification of glycosylation sites in protein sequences becomes increasingly important in the post-genomic era. A new encoding scheme was employed to improve the prediction of mucin-type O-glycosylation sites in mammalian proteins. Results A new protein bioinformatics tool, CKSAAP_OGlySite, was developed to predict mucin-type O-glycosylation serine/threonine (S/T sites in mammalian proteins. Using the composition of k-spaced amino acid pairs (CKSAAP based encoding scheme, the proposed method was trained and tested in a new and stringent O-glycosylation dataset with the assistance of Support Vector Machine (SVM. When the ratio of O-glycosylation to non-glycosylation sites in training datasets was set as 1:1, 10-fold cross-validation tests showed that the proposed method yielded a high accuracy of 83.1% and 81.4% in predicting O-glycosylated S and T sites, respectively. Based on the same datasets, CKSAAP_OGlySite resulted in a higher accuracy than the conventional binary encoding based method (about +5.0%. When trained and tested in 1:5 datasets, the CKSAAP encoding showed a more significant improvement than the binary encoding. We also merged the training datasets of S and T sites and integrated the prediction of S and T sites into one single predictor (i.e. S+T predictor. Either in 1:1 or 1:5 datasets, the performance of this S+T predictor was always slightly better than those predictors where S and T sites were independently predicted, suggesting that the molecular recognition of O-glycosylated S/T sites seems to be similar and the increase of the S+T predictor's accuracy may be a result of expanded training datasets. Moreover, CKSAAP_OGlySite was also shown to have better performance when benchmarked against two existing predictors. Conclusion Because of CKSAAP

Study Bioprospecting of Medicinal Plant Extracts of the Semiarid Northeast: Contribution to the Control of Oral Microorganisms

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Maria Suênia P. Silva

2012-01-01

Full Text Available Dental pathologies can be caused by plaque-forming bacteria and yeast, which reside in the oral cavity. The bacteria growing in dental plaque, a naturally occurring biofilm, display increased resistance to antimicrobial agents. The objective was the evaluation of a preclinical assay of medicinal plants of the semiarid region from the northeast against oral pathogenic microorganism, aiming at bioprospecting a new product. The selection of plant material for this study was based on the ethnobotanical data on the traditional use of plants from the semiarid region. The thirty extracts were subjected to the determination of antibiofilm activity against gram-positive, gram-negative bacteria and yeast. The hydroalcoholic extract which showed positive antibiofilm activity against most of the microorganisms tested in agar diffusion assay was further tested for the determination of minimum inhibitory concentration (MIC and Bioassay with Artemia salina. Plant samples tested in this study exhibited good antibiofilm activity for the treatment of oral problems. The Schinopsis brasiliensis showed greater activity for Pseudomonas aeruginosa and Staphylococcus aureus, but toxicity against Artemia salina.

Protein N-glycosylation in eukaryotic microalgae and its impact on the production of nuclear expressed biopharmaceuticals

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Elodie eMathieu-Rivet

2014-07-01

Full Text Available Microalgae are currently used for the production of food compounds. Recently, few microalgae species have been investigated as potential biofactories for the production of biopharmaceuticals. Indeed in this context, microalgae are cheap, classified as Generally Recognized As Safe (GRAS organisms and can be grown easily. However, problems remain to be solved before any industrial production of microalgae-made biopharmaceuticals. Among them, post-translational modifications of the proteins need to be considered. Especially, N-glycosylation acquired by the secreted recombinant proteins is of major concern since most of the biopharmaceuticals are N-glycosylated and it is well recognized that glycosylation represent one of their critical quality attribute. Therefore, the evaluation of microalgae as alternative cell factory for biopharmaceutical productions thus requires to investigate their N-glycosylation capability in order to determine to what extend it differs from their human counterpart and to determine appropriate strategies for remodelling the microalgae glycosylation into human-compatible oligosaccharides. Here, we review the secreted recombinant proteins which have been successfully produced in microalgae. We also report on recent bioinformatics and biochemical data concerning the structure of glycans N-linked to proteins from various microalgae phyla and comment the consequences on the glycan engineering strategies that may be necessary to render those microalgae-made biopharmaceuticals compatible with human therapy.

A metagenomic approach to decipher the indigenous microbial communities of arsenic contaminated groundwater of Assam

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Saurav Das

2017-06-01

Full Text Available Metagenomic approach was used to understand the structural and functional diversity present in arsenic contaminated groundwater of the Ganges Brahmaputra Delta aquifer system. A metagene dataset (coded as TTGW1 of 89,171 sequences (totaling 125,449,864 base pairs with an average length of 1406 bps was annotated. About 74,478 sequences containing 101,948 predicted protein coding regions passed the quality control. Taxonomical classification revealed abundance of bacteria that accounted for 98.3% of the microbial population of the metagenome. Eukaryota had an abundance of 1.1% followed by archea that showed 0.4% abundance. In phylum based classification, Proteobacteria was dominant (62.6% followed by Bacteroidetes (11.7%, Planctomycetes (7.7%, Verrucomicrobia (5.6%, Actinobacteria (3.7% and Firmicutes (1.9%. The Clusters of Orthologous Groups (COGs analysis indicated that the protein regulating the metabolic functions constituted a high percentage (18,199 reads; 39.3% of the whole metagenome followed by the proteins regulating the cellular processes (22.3%. About 0.07% sequences of the whole metagenome were related to genes coding for arsenic resistant mechanisms. Nearly 50% sequences of these coded for the arsenate reductase enzyme (EC. 1.20.4.1, the dominant enzyme of ars operon. Proteins associated with iron acquisition and metabolism were coded by 2% of the metagenome as revealed through SEED analysis. Our study reveals the microbial diversity and provides an insight into the functional aspect of the genes that might play crucial role in arsenic geocycle in contaminated ground water of Assam.

Systems analysis of singly and multiply O-glycosylated peptides in the human serum glycoproteome via EThcD and HCD mass spectrometry.

Science.gov (United States)

Zhang, Yong; Xie, Xinfang; Zhao, Xinyuan; Tian, Fang; Lv, Jicheng; Ying, Wantao; Qian, Xiaohong

2018-01-06

Human serum has been intensively studied to identify biomarkers via global proteomic analysis. The altered O-glycoproteome is associated with human pathological state including cancer, inflammatory and degenerative diseases and is an attractive source of disease biomarkers. Because of the microheterogeneity and macroheterogeneity of O-glycosylation, site-specific O-glycosylation analysis in human serum is still challenging. Here, we developed a systematic strategy that combined multiple enzyme digestion, multidimensional separation for sample preparation and high-resolution tandem MS with Byonic software for intact O-glycopeptide characterization. We demonstrated that multiple enzyme digestion or multidimensional separation can make sample preparation more efficient and that EThcD is not only suitable for the identification of singly O-glycosylated peptides (50.3%) but also doubly (21.2%) and triply (28.5%) O-glycosylated peptides. Totally, with the strict scoring criteria, 499 non-redundant intact O-glycopeptides, 173 O-glycosylation sites and 6 types of O-glycans originating from 49 O-glycoprotein groups were identified in human serum, including 121 novel O-glycosylation sites. Currently, this is the largest data set of site-specific native O-glycoproteome from human serum samples. We expect that the strategies developed by this study will facilitate in-depth analyses of native O-glycoproteomes in human serum and provide opportunities to understand the functional roles of protein O-glycosylation in human health and diseases. The altered O-glycoproteome is associated with human pathological state and is an attractive source of disease biomarkers. However, site-specific O-glycosylation analysis is challenging because of the microheterogeneity (different glycoforms attached to one glycosylation site) and macroheterogeneity (site occupancy) of O-glycosylation. In this work, we developed a systematic strategy for intact O-glycopeptide characterization. This study took

A Statistical Framework for the Functional Analysis of Metagenomes

Energy Technology Data Exchange (ETDEWEB)

Sharon, Itai; Pati, Amrita; Markowitz, Victor; Pinter, Ron Y.

2008-10-01

Metagenomic studies consider the genetic makeup of microbial communities as a whole, rather than their individual member organisms. The functional and metabolic potential of microbial communities can be analyzed by comparing the relative abundance of gene families in their collective genomic sequences (metagenome) under different conditions. Such comparisons require accurate estimation of gene family frequencies. They present a statistical framework for assessing these frequencies based on the Lander-Waterman theory developed originally for Whole Genome Shotgun (WGS) sequencing projects. They also provide a novel method for assessing the reliability of the estimations which can be used for removing seemingly unreliable measurements. They tested their method on a wide range of datasets, including simulated genomes and real WGS data from sequencing projects of whole genomes. Results suggest that their framework corrects inherent biases in accepted methods and provides a good approximation to the true statistics of gene families in WGS projects.

Construction and Screening of Marine Metagenomic Large Insert Libraries.

Science.gov (United States)

Weiland-Bräuer, Nancy; Langfeldt, Daniela; Schmitz, Ruth A

2017-01-01

The marine environment covers more than 70 % of the world's surface. Marine microbial communities are highly diverse and have evolved during extended evolutionary processes of physiological adaptations under the influence of a variety of ecological conditions and selection pressures. They harbor an enormous diversity of microbes with still unknown and probably new physiological characteristics. In the past, marine microbes, mostly bacteria of microbial consortia attached to marine tissues of multicellular organisms, have proven to be a rich source of highly potent bioactive compounds, which represent a considerable number of drug candidates. However, to date, the biodiversity of marine microbes and the versatility of their bioactive compounds and metabolites have not been fully explored. This chapter describes sampling in the marine environment, construction of metagenomic large insert libraries from marine habitats, and exemplarily one function based screen of metagenomic clones for identification of quorum quenching activities.

Glycosylation profiles of therapeutic antibody pharmaceuticals.

Science.gov (United States)

Wacker, Christoph; Berger, Christoph N; Girard, Philippe; Meier, Roger

2011-11-01

Recombinant antibodies specific for human targets are often used as therapeutics and represent a major class of drug products. Their therapeutic efficacy depends on the formation of antibody complexes resulting in the elimination of a target molecule or the modulation of specific signalling pathways. The physiological effects of antibody therapeutics are known to depend on the structural characteristics of the antibody molecule, specifically on the glycosylation which is the result of posttranslational modifications. Hence, production of therapeutic antibodies with a defined and consistent glycoform profile is needed which still remains a considerable challenge to the biopharmaceutical industry. To provide an insight into the industries capability to control their manufacturing process and to provide antibodies of highest quality, we conducted a market surveillance study and compared major oligosaccharide profiles of a number of monoclonal antibody pharmaceuticals sampled on the Swiss market. Product lot-to-lot variability was found to be generally low, suggesting that a majority of manufacturers have implemented high quality standards in their production processes. However, proportions of G0, G1 and G2 core-fucosylated chains derived from different products varied considerably and showed a bias towards the immature agalactosidated G0 form. Interestingly, differences in glycosylation caused by the production cell type seem to be of less importance compared with process related parameters such as cell growth. Copyright © 2011 Elsevier B.V. All rights reserved.

High abundance of Serine/Threonine-rich regions predicted to be hyper-O-glycosylated in the secretory proteins coded by eight fungal genomes

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González Mario

2012-09-01

Full Text Available Abstract Background O-glycosylation of secretory proteins has been found to be an important factor in fungal biology and virulence. It consists in the addition of short glycosidic chains to Ser or Thr residues in the protein backbone via O-glycosidic bonds. Secretory proteins in fungi frequently display Ser/Thr rich regions that could be sites of extensive O-glycosylation. We have analyzed in silico the complete sets of putatively secretory proteins coded by eight fungal genomes (Botrytis cinerea, Magnaporthe grisea, Sclerotinia sclerotiorum, Ustilago maydis, Aspergillus nidulans, Neurospora crassa, Trichoderma reesei, and Saccharomyces cerevisiae in search of Ser/Thr-rich regions as well as regions predicted to be highly O-glycosylated by NetOGlyc (http://www.cbs.dtu.dk. Results By comparison with experimental data, NetOGlyc was found to overestimate the number of O-glycosylation sites in fungi by a factor of 1.5, but to be quite reliable in the prediction of highly O-glycosylated regions. About half of secretory proteins have at least one Ser/Thr-rich region, with a Ser/Thr content of at least 40% over an average length of 40 amino acids. Most secretory proteins in filamentous fungi were predicted to be O-glycosylated, sometimes in dozens or even hundreds of sites. Residues predicted to be O-glycosylated have a tendency to be grouped together forming hyper-O-glycosylated regions of varying length. Conclusions About one fourth of secretory fungal proteins were predicted to have at least one hyper-O-glycosylated region, which consists of 45 amino acids on average and displays at least one O-glycosylated Ser or Thr every four residues. These putative highly O-glycosylated regions can be found anywhere along the proteins but have a slight tendency to be at either one of the two ends.

Enzymatic Glycosylation of Small Molecules: Challenging Substrates Require Tailored Catalysts

Czech Academy of Sciences Publication Activity Database

Desmet, T.; Soetaert, W.; Bojarová, Pavla; Křen, Vladimír; Dijkhuizen, L.; Eastwick-Field, V.; Schiller, A.

2012-01-01

Roč. 18, č. 35 (2012), s. 10786-10801 ISSN 0947-6539 Institutional support: RVO:61388971 Keywords : acceptor specificity * enzyme engineering * glycosylation Subject RIV: CE - Biochemistry Impact factor: 5.831, year: 2012

The S-Layer Glycoprotein of the Crenarchaeote Sulfolobus acidocaldarius Is Glycosylated at Multiple Sites with Chitobiose-Linked N-Glycans

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Elham Peyfoon

2010-01-01

Full Text Available Glycosylation of the S-layer of the crenarchaea Sulfolobus acidocaldarius has been investigated using glycoproteomic methodologies. The mature protein is predicted to contain 31 N-glycosylation consensus sites with approximately one third being found in the C-terminal domain spanning residues L1004-Q1395. Since this domain is rich in Lys and Arg and therefore relatively tractable to glycoproteomic analysis, this study has focused on mapping its N-glycosylation. Our analysis identified nine of the 11 consensus sequence sites, and all were found to be glycosylated. This constitutes a remarkably high glycosylation density in the C-terminal domain averaging one site for each stretch of 30–40 residues. Each of the glycosylation sites observed was shown to be modified with a heterogeneous family of glycans, with the largest having a composition Glc1Man2GlcNAc2 plus 6-sulfoquinovose (QuiS, consistent with the tribranched hexasaccharide previously reported in the cytochrome b558/566 of S. acidocaldarius. S. acidocaldarius is the only archaeal species whose N-glycans are known to be linked via the chitobiose core disaccharide that characterises the N-linked glycans of Eukarya.

Glycosylation-mediated phenylpropanoid partitioning in Populus tremuloides cell cultures

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Babst Benjamin A

2009-12-01

Full Text Available Abstract Background Phenylpropanoid-derived phenolic glycosides (PGs and condensed tannins (CTs comprise large, multi-purpose non-structural carbon sinks in Populus. A negative correlation between PG and CT concentrations has been observed in several studies. However, the molecular mechanism underlying the relationship is not known. Results Populus cell cultures produce CTs but not PGs under normal conditions. Feeding salicyl alcohol resulted in accumulation of salicins, the simplest PG, in the cells, but not higher-order PGs. Salicin accrual reflected the stimulation of a glycosylation response which altered a number of metabolic activities. We utilized this suspension cell feeding system as a model for analyzing the possible role of glycosylation in regulating the metabolic competition between PG formation, CT synthesis and growth. Cells accumulated salicins in a dose-dependent manner following salicyl alcohol feeding. Higher feeding levels led to a decrease in cellular CT concentrations (at 5 or 10 mM, and a negative effect on cell growth (at 10 mM. The competition between salicin and CT formation was reciprocal, and depended on the metabolic status of the cells. We analyzed gene expression changes between controls and cells fed with 5 mM salicyl alcohol for 48 hr, a time point when salicin accumulation was near maximum and CT synthesis was reduced, with no effect on growth. Several stress-responsive genes were up-regulated, suggestive of a general stress response in the fed cells. Salicyl alcohol feeding also induced expression of genes associated with sucrose catabolism, glycolysis and the Krebs cycle. Transcript levels of phenylalanine ammonia lyase and most of the flavonoid pathway genes were reduced, consistent with down-regulated CT synthesis. Conclusions Exogenous salicyl alcohol was readily glycosylated in Populus cell cultures, a process that altered sugar utilization and phenolic partitioning in the cells. Using this system, we

High definition for systems biology of microbial communities: metagenomics gets genome-centric and strain-resolved.

Science.gov (United States)

Turaev, Dmitrij; Rattei, Thomas

2016-06-01

The systems biology of microbial communities, organismal communities inhabiting all ecological niches on earth, has in recent years been strongly facilitated by the rapid development of experimental, sequencing and data analysis methods. Novel experimental approaches and binning methods in metagenomics render the semi-automatic reconstructions of near-complete genomes of uncultivable bacteria possible, while advances in high-resolution amplicon analysis allow for efficient and less biased taxonomic community characterization. This will also facilitate predictive modeling approaches, hitherto limited by the low resolution of metagenomic data. In this review, we pinpoint the most promising current developments in metagenomics. They facilitate microbial systems biology towards a systemic understanding of mechanisms in microbial communities with scopes of application in many areas of our daily life. Copyright © 2016 Elsevier Ltd. All rights reserved.

Glycosyl-Nucleolipids as New Bioinspired Amphiphiles

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Philippe Barthélémy

2013-09-01

Full Text Available Four new Glycosyl-NucleoLipid (GNL analogs featuring either a single fluorocarbon or double hydrocarbon chains were synthesized in good yields from azido thymidine as starting material. Physicochemical studies (surface tension measurements, differential scanning calorimetry indicate that hydroxybutanamide-based GNLs feature endothermic phase transition temperatures like the previously reported double chain glycerol-based GNLs. The second generation of GNFs featuring a free nucleobase reported here presents a better surface activity (lower glim compared to the first generation of GNFs.

A model to explain plant growth promotion traits: a multivariate analysis of 2,211 bacterial isolates.

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Pedro Beschoren da Costa

Full Text Available Plant growth-promoting bacteria can greatly assist sustainable farming by improving plant health and biomass while reducing fertilizer use. The plant-microorganism-environment interaction is an open and complex system, and despite the active research in the area, patterns in root ecology are elusive. Here, we simultaneously analyzed the plant growth-promoting bacteria datasets from seven independent studies that shared a methodology for bioprospection and phenotype screening. The soil richness of the isolate's origin was classified by a Principal Component Analysis. A Categorical Principal Component Analysis was used to classify the soil richness according to isolate's indolic compound production, siderophores production and phosphate solubilization abilities, and bacterial genera composition. Multiple patterns and relationships were found and verified with nonparametric hypothesis testing. Including niche colonization in the analysis, we proposed a model to explain the expression of bacterial plant growth-promoting traits according to the soil nutritional status. Our model shows that plants favor interaction with growth hormone producers under rich nutrient conditions but favor nutrient solubilizers under poor conditions. We also performed several comparisons among the different genera, highlighting interesting ecological interactions and limitations. Our model could be used to direct plant growth-promoting bacteria bioprospection and metagenomic sampling.

Variability in metagenomic samples from the Puget Sound: Relationship to temporal and anthropogenic impacts.

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James C Wallace

Full Text Available Whole-metagenome sequencing (WMS has emerged as a powerful tool to assess potential public health risks in marine environments by measuring changes in microbial community structure and function in uncultured bacteria. In addition to monitoring public health risks such as antibiotic resistance determinants, it is essential to measure predictors of microbial variation in order to identify natural versus anthropogenic factors as well as to evaluate reproducibility of metagenomic measurements.This study expands our previous metagenomic characterization of Puget Sound by sampling new nearshore environments including the Duwamish River, an EPA superfund site, and the Hood Canal, an area characterized by highly variable oxygen levels. We also resampled a wastewater treatment plant, nearshore and open ocean sites introducing a longitudinal component measuring seasonal and locational variations and establishing metagenomics sampling reproducibility. Microbial composition from samples collected in the open sound were highly similar within the same season and location across different years, while nearshore samples revealed multi-fold seasonal variation in microbial composition and diversity. Comparisons with recently sequenced predominant marine bacterial genomes helped provide much greater species level taxonomic detail compared to our previous study. Antibiotic resistance determinants and pollution and detoxification indicators largely grouped by location showing minor seasonal differences. Metal resistance, oxidative stress and detoxification systems showed no increase in samples proximal to an EPA superfund site indicating a lack of ecosystem adaptation to anthropogenic impacts. Taxonomic analysis of common sewage influent families showed a surprising similarity between wastewater treatment plant and open sound samples suggesting a low-level but pervasive sewage influent signature in Puget Sound surface waters. Our study shows reproducibility of

Glycosylation in HIV-1 envelope glycoprotein and its biological implications

KAUST Repository

Ho, Yung Shwen; Saksena, Nitin K.

2013-01-01

architecture, also controls intra- and inter-clade genetic variations. Discerning intra- and inter-clade glycosylation variations could therefore yield important information for understanding the molecular and biological differences between HIV clades and may

UGT74AN1, a Permissive Glycosyltransferase from Asclepias curassavica for the Regiospecific Steroid 3-O-Glycosylation.

Science.gov (United States)

Wen, Chao; Huang, Wei; Zhu, Xue-Lin; Li, Xiao-San; Zhang, Fan; Jiang, Ren-Wang

2018-02-02

A permissive steroid glycosyltransferase (UGT74AN1) from Asclepias curassavica exhibited robust capabilities for the regiospecific C3 glycosylation of cardiotonic steroids and C 21 steroid precursors, and unprecedented promiscuity toward 53 structurally diverse natural and unnatural compounds to form O-, N-, and S-glycosides, along with the catalytic reversibility for a one-pot transglycosylation reaction. These findings highlight UGT74AN1 as the first regiospecific catalyst for cardiotonic steroid C3 glycosylation and exhibit significant potential for glycosylation of diverse bioactive molecules in drug discovery.

Centrifuge: rapid and sensitive classification of metagenomic sequences.

Science.gov (United States)

Kim, Daehwan; Song, Li; Breitwieser, Florian P; Salzberg, Steven L

2016-12-01

Centrifuge is a novel microbial classification engine that enables rapid, accurate, and sensitive labeling of reads and quantification of species on desktop computers. The system uses an indexing scheme based on the Burrows-Wheeler transform (BWT) and the Ferragina-Manzini (FM) index, optimized specifically for the metagenomic classification problem. Centrifuge requires a relatively small index (4.2 GB for 4078 bacterial and 200 archaeal genomes) and classifies sequences at very high speed, allowing it to process the millions of reads from a typical high-throughput DNA sequencing run within a few minutes. Together, these advances enable timely and accurate analysis of large metagenomics data sets on conventional desktop computers. Because of its space-optimized indexing schemes, Centrifuge also makes it possible to index the entire NCBI nonredundant nucleotide sequence database (a total of 109 billion bases) with an index size of 69 GB, in contrast to k-mer-based indexing schemes, which require far more extensive space. © 2016 Kim et al.; Published by Cold Spring Harbor Laboratory Press.

The interdomain flexible linker of the polypeptide GalNAc transferases dictates their long-range glycosylation preferences

DEFF Research Database (Denmark)

Rivas, Matilde De Las; Lira-Navarrete, Erandi; Daniel, Earnest James Paul

2017-01-01

The polypeptide GalNAc-transferases (GalNAc-Ts), that initiate mucin-type O-glycosylation, consist of a catalytic and a lectin domain connected by a flexible linker. In addition to recognizing polypeptide sequence, the GalNAc-Ts exhibit unique long-range N- A nd/or C-terminal prior glycosylation ...

Bacterial tag encoded FLX titanium amplicon pyrosequencing (bTEFAP based assessment of prokaryotic diversity in metagenome of Lonar soda lake, India

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Pravin Dudhagara

2015-06-01

Full Text Available Bacterial diversity and archaeal diversity in metagenome of the Lonar soda lake sediment were assessed by bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP. Metagenome comprised 5093 sequences with 2,531,282 bp and 53 ± 2% G + C content. Metagenome sequence data are available at NCBI under the Bioproject database with accession no. PRJNA218849. Metagenome sequence represented the presence of 83.1% bacterial and 10.5% archaeal origin. A total of 14 different bacteria demonstrating 57 species were recorded with dominating species like Coxiella burnetii (17%, Fibrobacter intestinalis (12% and Candidatus Cloacamonas acidaminovorans (11%. Occurrence of two archaeal phyla representing 24 species, among them Methanosaeta harundinacea (35%, Methanoculleus chikugoensis (12% and Methanolinea tarda (11% were dominating species. Significant presence of 11% sequences as an unclassified indicated the possibilities for unknown novel prokaryotes from the metagenome.

Encoding asymmetry of the N-glycosylation motif facilitates glycoprotein evolution.

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Ryan Williams

Full Text Available Protein N-glycosylation is found in all domains of life and has a conserved role in glycoprotein folding and stability. In animals, glycoproteins transit through the Golgi where the N-glycans are trimmed and rebuilt with sequences that bind lectins, an innovation that greatly increases structural diversity and redundancy of glycoprotein-lectin interaction at the cell surface. Here we ask whether the natural tension between increasing diversity (glycan-protein interactions and site multiplicity (backup and status quo might be revealed by a phylogenic examination of glycoproteins and NXS/T(X ≠ P N-glycosylation sites. Site loss is more likely by mutation at Asn encoded by two adenosine (A-rich codons, while site gain is more probable by generating Ser or Thr downstream of an existing Asn. Thus mutations produce sites at novel positions more frequently than the reversal of recently lost sites, and therefore more paths though sequence space are made available to natural selection. An intra-species comparison of secretory and cytosolic proteins revealed a departure from equilibrium in sequences one-mutation-away from NXS/T and in (A content, indicating strong selective pressures and exploration of N-glycosylation positions during vertebrate evolution. Furthermore, secretory proteins have evolved at rates proportional to N-glycosylation site number, indicating adaptive interactions between the N-glycans and underlying protein. Given the topology of the genetic code, mutation of (A is more often nonsynonomous, and Lys, another target of many PTMs, is also encoded by two (A-rich codons. An examination of acetyl-Lys sites in proteins indicated similar evolutionary dynamics, consistent with asymmetry of the target and recognition portions of modified sites. Our results suggest that encoding asymmetry is an ancient mechanism of evolvability that increases diversity and experimentation with PTM site positions. Strong selective pressures on PTMs may have

Bioprospection in Brazil and Mexico, a new El Dorado?: Between the Instability of Practices and the Permanence of Representations

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Geoffroy Filoche

2011-01-01

Full Text Available Because of their exceptional biodiversity, Brazil and Mexico seemed to be the ideal candidates for the valorization of their genetic resources, as promoted by the Convention on Biological Diversity (CBD. But almost twenty years after the Rio Conference, the actual results of this valorization are disappointing. It can be explained either by the slowness in adopting domestic laws translating the CBD or by the difficulties in controlling and regulating very complex and unstable practices and objects. This leads to put into question the pertinence of the CDB framework, along with its categories and mechanisms. In spite of the relative obsolescence of this framework and the emergence of new environmental issues, which appear as more important, the question of the access to genetic resources and their exploitation through contracts of bioprospection is still being debated because of the various symbolic issues at stake, such as national sovereignty and the defense of biological and cultural diversity.

Glycosylated yellow laccases of the basidiomycete Stropharia aeruginosa.

Science.gov (United States)

Daroch, Maurycy; Houghton, Catharine A; Moore, Jonathan K; Wilkinson, Mark C; Carnell, Andrew J; Bates, Andrew D; Iwanejko, Lesley A

2014-05-10

Here we describe the identification, purification and characterisation of glycosylated yellow laccase proteins from the basidiomycete fungus Stropharia aeruginosa. Biochemical characterisation of two yellow laccases, Yel1p and Yel3p, show that they are both secreted, monomeric, N-glycosylated proteins of molecular weight around 55kDa with substrate specificities typical of laccases, but lacking the absorption band at 612nm typical of the blue laccase proteins. Low coverage, high throughput 454 transcriptome sequencing in combination with inverse-PCR was used to identify cDNA sequences. One of the cDNA sequences has been assigned to the Yel1p protein on the basis of identity between the translated protein sequence and the peptide data from the purified protein, and the full length gene sequence has been obtained. Biochemical properties, substrate specificities and protein sequence data have been used to discuss the unusual spectroscopic properties of S. aeruginosa proteins in the context of recent theories about the differences between yellow and blue laccases. Copyright © 2014 Elsevier Inc. All rights reserved.

N-glycosylation of the β2 adrenergic receptor regulates receptor function by modulating dimerization.

Science.gov (United States)

Li, Xiaona; Zhou, Mang; Huang, Wei; Yang, Huaiyu

2017-07-01

N-glycosylation is a common post-translational modification of G-protein-coupled receptors (GPCRs). However, it remains unknown how N-glycosylation affects GPCR signaling. β 2 adrenergic receptor (β 2 AR) has three N-glycosylation sites: Asn6, Asn15 at the N-terminus, and Asn187 at the second extracellular loop (ECL2). Here, we show that deletion of the N-glycan did not affect receptor expression and ligand binding. Deletion of the N-glycan at the N-terminus rather than Asn187 showed decreased effects on isoproterenol-promoted G-protein-dependent signaling, β-arrestin2 recruitment, and receptor internalization. Both N6Q and N15Q showed decreased receptor dimerization, while N187Q did not influence receptor dimerization. As decreased β 2 AR homodimer accompanied with reduced efficiency for receptor function, we proposed that the N-glycosylation of β 2 AR regulated receptor function by influencing receptor dimerization. To verify this hypothesis, we further paid attention to the residues at the dimerization interface. Studies of Lys60 and Glu338, two residues at the receptor dimerization interface, exhibited that the K60A/E338A showed decreased β 2 AR dimerization and its effects on receptor signaling were similar to N6Q and N15Q, which further supported the importance of receptor dimerization for receptor function. This work provides new insights into the relationship among glycosylation, dimerization, and function of GPCRs. Peptide-N-glycosidase F (PNGase F, EC 3.2.2.11); endo-β-N-acetylglucosaminidase A (Endo-A, EC 3.2.1.96). © 2017 Federation of European Biochemical Societies.

Altered protein glycosylation predicts Alzheimer's disease and modulates its pathology in disease model Drosophila.

Science.gov (United States)

Frenkel-Pinter, Moran; Stempler, Shiri; Tal-Mazaki, Sharon; Losev, Yelena; Singh-Anand, Avnika; Escobar-Álvarez, Daniela; Lezmy, Jonathan; Gazit, Ehud; Ruppin, Eytan; Segal, Daniel

2017-08-01

The pathological hallmarks of Alzheimer's disease (AD) are pathogenic oligomers and fibrils of misfolded amyloidogenic proteins (e.g., β-amyloid and hyper-phosphorylated tau in AD), which cause progressive loss of neurons in the brain and nervous system. Although deviations from normal protein glycosylation have been documented in AD, their role in disease pathology has been barely explored. Here our analysis of available expression data sets indicates that many glycosylation-related genes are differentially expressed in brains of AD patients compared with healthy controls. The robust differences found enabled us to predict the occurrence of AD with remarkable accuracy in a test cohort and identify a set of key genes whose expression determines this classification. We then studied in vivo the effect of reducing expression of homologs of 6 of these genes in transgenic Drosophila overexpressing human tau, a well-established invertebrate AD model. These experiments have led to the identification of glycosylation genes that may augment or ameliorate tauopathy phenotypes. Our results indicate that OstDelta, l(2)not and beta4GalT7 are tauopathy suppressors, whereas pgnat5 and CG33303 are enhancers, of tauopathy. These results suggest that specific alterations in protein glycosylation may play a causal role in AD etiology. Copyright © 2017 Elsevier Inc. All rights reserved.

A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation

DEFF Research Database (Denmark)

Hägglund, Per; Bunkenborg, Jakob; Elortza, Felix

2004-01-01

remains linked to the asparagine residue. The removal of the major part of the glycan simplifies the MS/MS fragment ion spectra of glycopeptides, while the remaining GlcNAc residue enables unambiguous assignment of the glycosylation site together with the amino acid sequence. We first tested our approach...

Re-Analysis of Metagenomic Sequences from Acute Flaccidmyelitis Patients Reveals Alternatives to Enterovirus D68 Infection

Science.gov (United States)

2015-07-13

caused in some cases by infection with enterovirus D68. We found that among the patients whose symptoms were previously attributed to enterovirus D68...distribution is unlimited. Re-analysis of metagenomic sequences from acute flaccidmyelitis patients reveals alternatives to enterovirus D68...Street Baltimore, MD 21218 -2685 ABSTRACT Re-analysis of metagenomic sequences from acute flaccidmyelitis patients reveals alternatives to enterovirus

Preventing E-cadherin aberrant N-glycosylation at Asn-554 improves its critical function in gastric cancer

Science.gov (United States)

Carvalho, S; Catarino, TA; Dias, AM; Kato, M; Almeida, A; Hessling, B; Figueiredo, J; Gärtner, F; Sanches, JM; Ruppert, T; Miyoshi, E; Pierce, M; Carneiro, F; Kolarich, D; Seruca, R; Yamaguchi, Y; Taniguchi, N; Reis, CA; Pinho, SS

2016-01-01

E-cadherin is a central molecule in the process of gastric carcinogenesis and its posttranslational modifications by N-glycosylation have been described to induce a deleterious effect on cell adhesion associated with tumor cell invasion. However, the role that site-specific glycosylation of E-cadherin has in its defective function in gastric cancer cells needs to be determined. Using transgenic mice models and human clinical samples, we demonstrated that N-acetylglucosaminyltransferase V (GnT-V)-mediated glycosylation causes an abnormal pattern of E-cadherin expression in the gastric mucosa. In vitro models further indicated that, among the four potential N-glycosylation sites of E-cadherin, Asn-554 is the key site that is selectively modified with β1,6 GlcNAc-branched N-glycans catalyzed by GnT-V. This aberrant glycan modification on this specific asparagine site of E-cadherin was demonstrated to affect its critical functions in gastric cancer cells by affecting E-cadherin cellular localization, cis-dimer formation, molecular assembly and stability of the adherens junctions and cell–cell aggregation, which was further observed in human gastric carcinomas. Interestingly, manipulating this site-specific glycosylation, by preventing Asn-554 from receiving the deleterious branched structures, either by a mutation or by silencing GnT-V, resulted in a protective effect on E-cadherin, precluding its functional dysregulation and contributing to tumor suppression. PMID:26189796

Diverse circovirus-like genome architectures revealed by environmental metagenomics.

Science.gov (United States)

Rosario, Karyna; Duffy, Siobain; Breitbart, Mya

2009-10-01

Single-stranded DNA (ssDNA) viruses with circular genomes are the smallest viruses known to infect eukaryotes. The present study identified 10 novel genomes similar to ssDNA circoviruses through data-mining of public viral metagenomes. The metagenomic libraries included samples from reclaimed water and three different marine environments (Chesapeake Bay, British Columbia coastal waters and Sargasso Sea). All the genomes have similarities to the replication (Rep) protein of circoviruses; however, only half have genomic features consistent with known circoviruses. Some of the genomes exhibit a mixture of genomic features associated with different families of ssDNA viruses (i.e. circoviruses, geminiviruses and parvoviruses). Unique genome architectures and phylogenetic analysis of the Rep protein suggest that these viruses belong to novel genera and/or families. Investigating the complex community of ssDNA viruses in the environment can lead to the discovery of divergent species and help elucidate evolutionary links between ssDNA viruses.

Quantification of the N-glycosylated secretome by super-SILAC during breast cancer progression and in human blood Samples

DEFF Research Database (Denmark)

Boersema, P.J.; Geiger, T.; Wiśniewski, J.R.

2013-01-01

Cells secrete a large number of proteins to communicate with their surroundings. Furthermore, plasma membrane proteins and intracellular proteins can be released into the extracellular space by regulated or non-regulated processes. Here, we profiled the supernatant of 11 cell lines....... In total, 1398 unique N-glycosylation sites were identified and quantified. Enriching for N-glycosylated peptides focused the analysis on classically secreted and membrane proteins. N-glycosylated secretome profiles correctly clustered the different cell lines to their respective cancer stage, suggesting...

[Proteins modified in the nonenzymatically glycosylation reaction (AGE-proteins)--new markers for diabetes?].

Science.gov (United States)

Zdrojewicz, Z; Januszewski, A; Kwiatkowska, D

1994-01-01

Paper present a recent review on the formation and clinical significance of advanced glycosylation end products, produced in nonenzymatically glycosylation, called Maillard reaction. The special attention was paid to AGEs role in diabetic and aging processes. Instant of occurring of AGEs in circulation or increase of AGE receptor concentration are many years faster than clinical pathology of vessels, nervous or kidneys connect with diabetes or aging. May be in the future it will be possible to decrease the consequence of Maillard reaction by using pharmacology drugs.

Glycosylation intermediates studied using low temperature ¹H- and ¹⁹F-DOSY NMR

DEFF Research Database (Denmark)

Qiao, Yan; Ge, Wenzhi; Jia, Lingyu

2016-01-01

Low temperature 1H- and 19F-DOSY have been used for analyzing reactive intermediates in glycosylation reactions, where a glycosyl trichloroacetimidate donor has been activated using different catalysts. The DOSY protocols have been optimized for low temperature experiments and provided new insight...

Glycosyl azide-a novel substrate for enzymatic transgycosylations

Czech Academy of Sciences Publication Activity Database

Fialová, Pavla; Carmona, A. T.; Robina, I.; Ettrich, R.; Sedmera, Petr; Přikrylová, Věra; Hušáková, Lucie; Křen, Vladimír

2005-01-01

Roč. 46, - (2005), s. 8715-8718 ISSN 0040-4039 R&D Projects: GA ČR GA203/05/0172; GA MŠk OC D25.002 Grant - others:GA KONTAKT 1862/04 Institutional research plan: CEZ:AV0Z50200510 Keywords : enzyme catalysis * glycosyl azide * molecular modelling Subject RIV: EE - Microbiology, Virology Impact factor: 2.477, year: 2005

Bioprospecting of Marine Invertebrates for New Natural Products — A Chemical and Zoogeographical Perspective

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Ricardo Calado

2012-08-01

Full Text Available Bioprospecting for new marine natural products (NPs has increased significantly over the last decades, leading to an unprecedented discovery of new molecules. Marine invertebrates have been the most important source of these NPs, with researchers commonly targeting particular taxonomic groups, marine regions and/or molecules from specific chemical groups. The present review focuses on new NPs identified from marine invertebrates between 2000 and 2009, and performs a detailed analysis on: (1 the chemical groups of these NPs; (2 the association of particular chemical groups to specific marine invertebrate taxa; and (3 the yielding of molecules from the same chemical group from organisms occurring in a particular geographic region. Our survey revealed an increasing number of new terpenoids being discovered between 2000 and 2009, contrasting with the decreasing trend in the discovery of new alkaloids and aliphatic molecules. Overall, no particular association was identified between marine invertebrate taxa and chemical groups of new NPs. Nonetheless, it is worth noting that most NPs recorded from cnidarians and mollusks were terpenoids, while most NPs identified in echinoderms were aliphatic compounds or carbohydrates. The geographical trends observed in our study do not support the idea of particular chemical groups of new NPs being associated with marine invertebrates from any specific geographical region, as NPs from different chemical groups were commonly distributed worldwide.

GenomePeek—an online tool for prokaryotic genome and metagenome analysis

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Katelyn McNair

2015-06-01

Full Text Available As more and more prokaryotic sequencing takes place, a method to quickly and accurately analyze this data is needed. Previous tools are mainly designed for metagenomic analysis and have limitations; such as long runtimes and significant false positive error rates. The online tool GenomePeek (edwards.sdsu.edu/GenomePeek was developed to analyze both single genome and metagenome sequencing files, quickly and with low error rates. GenomePeek uses a sequence assembly approach where reads to a set of conserved genes are extracted, assembled and then aligned against the highly specific reference database. GenomePeek was found to be faster than traditional approaches while still keeping error rates low, as well as offering unique data visualization options.

Biochemical Importance of Glycosylation of Plasminogen Activator Inhibitor-1

DEFF Research Database (Denmark)

Gils, Ann; Pedersen, Katrine Egelund; Skottrup, Peter Durand

2003-01-01

The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous glycosyla...

Crystallization and preliminary crystallographic analysis of human glycosylated haemoglobin

International Nuclear Information System (INIS)

Syakhovich, Vitaly E.; Saraswathi, N. T.; Ruff, Marc; Bokut, Sergey B.; Moras, Dino

2006-01-01

Non enzymatic modification of haemoglobin by glucose plays an important role in diabetes pathogenesis. Here the purification, characterization and crystallization of human glycosylated haemoglobin are reported. Human glycosylated haemoglobin A 1C is a stable minor variant formed in vivo by post-translational modification of the main form of haemoglobin by glucose. Crystals of oxyHbA 1C were obtained using the hanging-drop vapour-diffusion method and PEG as precipitant. The diffraction pattern of the crystal extends to a resolution of 2.3 Å at 120 K. The crystals belong to space group C2, with unit-cell parameters a = 237.98, b = 59.27, c = 137.02 Å, α = 90.00, β = 125.40, γ = 90.00°. The presence of two and a half molecules per asymmetric unit gives a crystal volume per protein weight (V M ) of 9.70 Å 3 Da −1 and a solvent content of 49%

Crystallization and preliminary crystallographic analysis of human glycosylated haemoglobin

Energy Technology Data Exchange (ETDEWEB)

Syakhovich, Vitaly E. [Department of Biochemistry and Biophysics, International Sakharov Environmental University, Dolgobrodskaya St 23, 220009 Minsk (Belarus); Saraswathi, N. T.; Ruff, Marc, E-mail: ruff@igbmc.u-strasbg.fr [Département de Biologie et Génomique Structurales, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 Rue Laurent Fries, BP 10142, 67404 Illkirch (France); Bokut, Sergey B. [Department of Biochemistry and Biophysics, International Sakharov Environmental University, Dolgobrodskaya St 23, 220009 Minsk (Belarus); Moras, Dino [Département de Biologie et Génomique Structurales, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 Rue Laurent Fries, BP 10142, 67404 Illkirch (France); Department of Biochemistry and Biophysics, International Sakharov Environmental University, Dolgobrodskaya St 23, 220009 Minsk (Belarus)

2006-02-01

Non enzymatic modification of haemoglobin by glucose plays an important role in diabetes pathogenesis. Here the purification, characterization and crystallization of human glycosylated haemoglobin are reported. Human glycosylated haemoglobin A{sub 1C} is a stable minor variant formed in vivo by post-translational modification of the main form of haemoglobin by glucose. Crystals of oxyHbA{sub 1C} were obtained using the hanging-drop vapour-diffusion method and PEG as precipitant. The diffraction pattern of the crystal extends to a resolution of 2.3 Å at 120 K. The crystals belong to space group C2, with unit-cell parameters a = 237.98, b = 59.27, c = 137.02 Å, α = 90.00, β = 125.40, γ = 90.00°. The presence of two and a half molecules per asymmetric unit gives a crystal volume per protein weight (V{sub M}) of 9.70 Å{sup 3} Da{sup −1} and a solvent content of 49%.

An Improved Methodology to Overcome Key Issues in Human Fecal Metagenomic DNA Extraction

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Jitendra Kumar

2016-12-01

Full Text Available Microbes are ubiquitously distributed in nature, and recent culture-independent studies have highlighted the significance of gut microbiota in human health and disease. Fecal DNA is the primary source for the majority of human gut microbiome studies. However, further improvement is needed to obtain fecal metagenomic DNA with sufficient amount and good quality but low host genomic DNA contamination. In the current study, we demonstrate a quick, robust, unbiased, and cost-effective method for the isolation of high molecular weight (>23 kb metagenomic DNA (260/280 ratio >1.8 with a good yield (55.8 ± 3.8 ng/mg of feces. We also confirm that there is very low human genomic DNA contamination (eubacterial: human genomic DNA marker genes = 227.9:1 in the human feces. The newly-developed method robustly performs for fresh as well as stored fecal samples as demonstrated by 16S rRNA gene sequencing using 454 FLX+. Moreover, 16S rRNA gene analysis indicated that compared to other DNA extraction methods tested, the fecal metagenomic DNA isolated with current methodology retains species richness and does not show microbial diversity biases, which is further confirmed by qPCR with a known quantity of spike-in genomes. Overall, our data highlight a protocol with a balance between quality, amount, user-friendliness, and cost effectiveness for its suitability toward usage for culture-independent analysis of the human gut microbiome, which provides a robust solution to overcome key issues associated with fecal metagenomic DNA isolation in human gut microbiome studies.

Metagenome Fragment Classification Using -Mer Frequency Profiles

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Gail Rosen

2008-01-01

Full Text Available A vast amount of microbial sequencing data is being generated through large-scale projects in ecology, agriculture, and human health. Efficient high-throughput methods are needed to analyze the mass amounts of metagenomic data, all DNA present in an environmental sample. A major obstacle in metagenomics is the inability to obtain accuracy using technology that yields short reads. We construct the unique -mer frequency profiles of 635 microbial genomes publicly available as of February 2008. These profiles are used to train a naive Bayes classifier (NBC that can be used to identify the genome of any fragment. We show that our method is comparable to BLAST for small 25 bp fragments but does not have the ambiguity of BLAST's tied top scores. We demonstrate that this approach is scalable to identify any fragment from hundreds of genomes. It also performs quite well at the strain, species, and genera levels and achieves strain resolution despite classifying ubiquitous genomic fragments (gene and nongene regions. Cross-validation analysis demonstrates that species-accuracy achieves 90% for highly-represented species containing an average of 8 strains. We demonstrate that such a tool can be used on the Sargasso Sea dataset, and our analysis shows that NBC can be further enhanced.

Unravelling core microbial metabolisms in the hypersaline microbial mats of Shark Bay using high-throughput metagenomics

Energy Technology Data Exchange (ETDEWEB)

Ruvindy, Rendy; White III, Richard Allen; Neilan, Brett Anthony; Burns, Brendan Paul

2015-05-29

Modern microbial mats are potential analogues of some of Earth’s earliest ecosystems. Excellent examples can be found in Shark Bay, Australia, with mats of various morphologies. To further our understanding of the functional genetic potential of these complex microbial ecosystems, we conducted for the first time shotgun metagenomic analyses. We assembled metagenomic nextgeneration sequencing data to classify the taxonomic and metabolic potential across diverse morphologies of marine mats in Shark Bay. The microbial community across taxonomic classifications using protein-coding and small subunit rRNA genes directly extracted from the metagenomes suggests that three phyla Proteobacteria, Cyanobacteria and Bacteriodetes dominate all marine mats. However, the microbial community structure between Shark Bay and Highbourne Cay (Bahamas) marine systems appears to be distinct from each other. The metabolic potential (based on SEED subsystem classifications) of the Shark Bay and Highbourne Cay microbial communities were also distinct. Shark Bay metagenomes have a metabolic pathway profile consisting of both heterotrophic and photosynthetic pathways, whereas Highbourne Cay appears to be dominated almost exclusively by photosynthetic pathways. Alternative non-rubisco-based carbon metabolism including reductive TCA cycle and 3-hydroxypropionate/4-hydroxybutyrate pathways is highly represented in Shark Bay metagenomes while not represented in Highbourne Cay microbial mats or any other mat forming ecosystems investigated to date. Potentially novel aspects of nitrogen cycling were also observed, as well as putative heavy metal cycling (arsenic, mercury, copper and cadmium). Finally, archaea are highly represented in Shark Bay and may have critical roles in overall ecosystem function in these modern microbial mats.

Technical Report: Benchmarking for Quasispecies Abundance Inference with Confidence Intervals from Metagenomic Sequence Data

Energy Technology Data Exchange (ETDEWEB)

McLoughlin, K. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

2016-01-22

The software application “MetaQuant” was developed by our group at Lawrence Livermore National Laboratory (LLNL). It is designed to profile microbial populations in a sample using data from whole-genome shotgun (WGS) metagenomic DNA sequencing. Several other metagenomic profiling applications have been described in the literature. We ran a series of benchmark tests to compare the performance of MetaQuant against that of a few existing profiling tools, using real and simulated sequence datasets. This report describes our benchmarking procedure and results.

Metagenomic investigation of the microbial diversity in a chrysotile asbestos mine pit pond, Lowell, Vermont, USA

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Heather E. Driscoll

2016-12-01

Full Text Available Here we report on a metagenomics investigation of the microbial diversity in a serpentine-hosted aquatic habitat created by chrysotile asbestos mining activity at the Vermont Asbestos Group (VAG Mine in northern Vermont, USA. The now-abandoned VAG Mine on Belvidere Mountain in the towns of Eden and Lowell includes three open-pit quarries, a flooded pit, mill buildings, roads, and >26 million metric tons of eroding mine waste that contribute alkaline mine drainage to the surrounding watershed. Metagenomes and water chemistry originated from aquatic samples taken at three depths (0.5 m, 3.5 m, and 25 m along the water column at three distinct, offshore sites within the mine's flooded pit (near 44°46′00.7673″, −72°31′36.2699″; UTM NAD 83 Zone 18 T 0695720 E, 4960030 N. Whole metagenome shotgun Illumina paired-end sequences were quality trimmed and analyzed based on a translated nucleotide search of NCBI-NR protein database and lowest common ancestor taxonomic assignments. Our results show strata within the pit pond water column can be distinguished by taxonomic composition and distribution, pH, temperature, conductivity, light intensity, and concentrations of dissolved oxygen. At the phylum level, metagenomes from 0.5 m and 3.5 m contained a similar distribution of taxa and were dominated by Actinobacteria (46% and 53% of reads, respectively, Proteobacteria (45% and 38%, respectively, and Bacteroidetes (7% in both. The metagenomes from 25 m showed a greater diversity of phyla and a different distribution of reads than the two upper strata: Proteobacteria (60%, Actinobacteria (18%, Planctomycetes, (10%, Bacteroidetes (5% and Cyanobacteria (2.5%, Armatimonadetes (<1%, Verrucomicrobia (<1%, Firmicutes (<1%, and Nitrospirae (<1%. Raw metagenome sequence data from each sample reside in NCBI's Short Read Archive (SRA ID: SRP056095 and are accessible through NCBI BioProject PRJNA277916.

Detection of site specific glycosylation in proteins using flow cytometry†

Science.gov (United States)

Jayakumar, Deepak; Marathe, Dhananjay D.; Neelamegham, Sriram

2009-01-01

We tested the possibility that it is possible to express unique peptide probes on cell surfaces and detect site-specific glycosylation on these peptides using flow cytometry. Such development can enhance the application of flow cytometry to detect and quantify post-translational modifications in proteins. To this end, the N-terminal section of the human leukocyte glycoprotein PSGL-1 (P-selectin glycoprotein ligand-1) was modified to contain a poly-histidine tag followed by a proteolytic cleavage site. Amino acids preceding the cleavage site have a single O-linked glycosylation site. The recombinant protein called PSGL-1 (HT) was expressed on the surface of two mammalian cell lines, CHO and HL-60, using a lentiviral delivery approach. Results demonstrate that the N-terminal portion of PSGL-1 (HT) can be released from these cells by protease, and the resulting peptide can be readily captured and detected using cytometry-bead assays. Using this strategy, the peptide was immunoprecipitated onto beads bearing mAbs against either the poly-histidine sequence or the human PSGL-1. The carbohydrate epitope associated with the released peptide was detected using HECA-452 and CSLEX-1, monoclonal antibodies that recognize the sialyl Lewis-X epitope. Finally, the peptide released from cells could be separated and enriched using nickel chelate beads. Overall, such an approach that combines recombinant protein expression with flow cytometry, may be useful to quantify changes in site-specific glycosylation for basic science and clinical applications. PMID:19735085

Optimization of a colorimetric assay for glycosylated human serum albumin

International Nuclear Information System (INIS)

Bohney, J.P.; Feldhoff, R.C.

1986-01-01

The thiobarbituric acid (TBA) assay has been used for several years to quantitate the amount of glucose which has been non-enzymatically linked to hemoglobin and other proteins. The ketoamine-protein adduct is converted to 5-hydroxymethylfurfural (HMF) by mild hydrolysis with oxalic acid. Reaction of HMF with TBA yields a colored product which has an absorbance maximum at 443 nm. Several modifications of the original procedure has been published, but none permit the unambiguous quantitation of glycosylated human serum albumin (glc-HSA). Problems relate to reagent preparation and stability, the time and temperature of hydrolysis, the choice of standards, and background color corrections. The authors have found that maximum color yield occurs after hydrolysis in an autoclave for 2 h. This increases the sensitivity 3-fold and cuts the assay time in half relative to hydrolysis for 4.5 h at 100 0 C. A NaBH 4 reduction of a parallel protein sample must be performed to correct for variable background color associated with different sample sources and amounts. HMF can be used as a standard, however, corrections must be made for HMF degradation. Fructose is a better standard, but HMF formation from fructose is faster than formation from glc-HSA. This may result in an underestimate of percent glycosylation. The best standard appears to be glc-HSA prepared with [ 3 H]glucose. It appears that with proper controls and standards the TBA assay can be used to determine actual rather than relative percent glycosylation

Loci associated with N-glycosylation of human immunoglobulin G show pleiotropy with autoimmune diseases and haematological cancers

NARCIS (Netherlands)

Lauc, G.; Huffman, J.E.; Pucic, M.; Zgaga, L.; Adamczyk, B.; Muzinic, A.; Novokmet, M.; Polasek, O.; Gornik, O.; Kristic, J.; Keser, T.; Vitart, V.; Scheijen, B.; Uh, H.W.; Molokhia, M.; Patrick, A.L.; McKeigue, P.; Kolcic, I.; Lukic, I.K.; Swann, O.; Leeuwen, F.N. van; Ruhaak, L.R.; Houwing-Duistermaat, J.J.; Slagboom, P.E.; Beekman, M.; Craen, A.J. de; Deelder, A.M.; Zeng, Q.; Wang, W.; Hastie, N.D.; Gyllensten, U.; Wilson, J.F.; Wuhrer, M.; Wright, A.F.; Rudd, P.M.; Hayward, C.; Aulchenko, Y.; Campbell, H.; Rudan, I.

2013-01-01

Glycosylation of immunoglobulin G (IgG) influences IgG effector function by modulating binding to Fc receptors. To identify genetic loci associated with IgG glycosylation, we quantitated N-linked IgG glycans using two approaches. After isolating IgG from human plasma, we performed 77 quantitative

Binning sequences using very sparse labels within a metagenome

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Halgamuge Saman K

2008-04-01

Full Text Available Abstract Background In metagenomic studies, a process called binning is necessary to assign contigs that belong to multiple species to their respective phylogenetic groups. Most of the current methods of binning, such as BLAST, k-mer and PhyloPythia, involve assigning sequence fragments by comparing sequence similarity or sequence composition with already-sequenced genomes that are still far from comprehensive. We propose a semi-supervised seeding method for binning that does not depend on knowledge of completed genomes. Instead, it extracts the flanking sequences of highly conserved 16S rRNA from the metagenome and uses them as seeds (labels to assign other reads based on their compositional similarity. Results The proposed seeding method is implemented on an unsupervised Growing Self-Organising Map (GSOM, and called Seeded GSOM (S-GSOM. We compared it with four well-known semi-supervised learning methods in a preliminary test, separating random-length prokaryotic sequence fragments sampled from the NCBI genome database. We identified the flanking sequences of the highly conserved 16S rRNA as suitable seeds that could be used to group the sequence fragments according to their species. S-GSOM showed superior performance compared to the semi-supervised methods tested. Additionally, S-GSOM may also be used to visually identify some species that do not have seeds. The proposed method was then applied to simulated metagenomic datasets using two different confidence threshold settings and compared with PhyloPythia, k-mer and BLAST. At the reference taxonomic level Order, S-GSOM outperformed all k-mer and BLAST results and showed comparable results with PhyloPythia for each of the corresponding confidence settings, where S-GSOM performed better than PhyloPythia in the ≥ 10 reads datasets and comparable in the ≥ 8 kb benchmark tests. Conclusion In the task of binning using semi-supervised learning methods, results indicate S-GSOM to be the best of

Metagenome-derived haloalkane dehalogenases with novel catalytic properties

Czech Academy of Sciences Publication Activity Database

Kotík, Michael; Vaňáček, P.; Kuňka, A.; Prokop, Z.; Dambrovský, J.

2017-01-01

Roč. 101, č. 16 (2017), s. 6385-6397 ISSN 0175-7598 R&D Projects: GA ČR GAP504/10/0137; GA MŠk(CZ) LM2015047; GA MŠk(CZ) LM2015055 Institutional support: RVO:61388971 Keywords : Haloalkane dehalogenase * Metagenomic DNA * Heterologous production Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 3.420, year: 2016

The GAAS metagenomic tool and its estimations of viral and microbial average genome size in four major biomes.

Science.gov (United States)

Angly, Florent E; Willner, Dana; Prieto-Davó, Alejandra; Edwards, Robert A; Schmieder, Robert; Vega-Thurber, Rebecca; Antonopoulos, Dionysios A; Barott, Katie; Cottrell, Matthew T; Desnues, Christelle; Dinsdale, Elizabeth A; Furlan, Mike; Haynes, Matthew; Henn, Matthew R; Hu, Yongfei; Kirchman, David L; McDole, Tracey; McPherson, John D; Meyer, Folker; Miller, R Michael; Mundt, Egbert; Naviaux, Robert K; Rodriguez-Mueller, Beltran; Stevens, Rick; Wegley, Linda; Zhang, Lixin; Zhu, Baoli; Rohwer, Forest

2009-12-01

Metagenomic studies characterize both the composition and diversity of uncultured viral and microbial communities. BLAST-based comparisons have typically been used for such analyses; however, sampling biases, high percentages of unknown sequences, and the use of arbitrary thresholds to find significant similarities can decrease the accuracy and validity of estimates. Here, we present Genome relative Abundance and Average Size (GAAS), a complete software package that provides improved estimates of community composition and average genome length for metagenomes in both textual and graphical formats. GAAS implements a novel methodology to control for sampling bias via length normalization, to adjust for multiple BLAST similarities by similarity weighting, and to select significant similarities using relative alignment lengths. In benchmark tests, the GAAS method was robust to both high percentages of unknown sequences and to variations in metagenomic sequence read lengths. Re-analysis of the Sargasso Sea virome using GAAS indicated that standard methodologies for metagenomic analysis may dramatically underestimate the abundance and importance of organisms with small genomes in environmental systems. Using GAAS, we conducted a meta-analysis of microbial and viral average genome lengths in over 150 metagenomes from four biomes to determine whether genome lengths vary consistently between and within biomes, and between microbial and viral communities from the same environment. Significant differences between biomes and within aquatic sub-biomes (oceans, hypersaline systems, freshwater, and microbialites) suggested that average genome length is a fundamental property of environments driven by factors at the sub-biome level. The behavior of paired viral and microbial metagenomes from the same environment indicated that microbial and viral average genome sizes are independent of each other, but indicative of community responses to stressors and environmental conditions.

Fast and accurate taxonomic assignments of metagenomic sequences using MetaBin.

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Vineet K Sharma

Full Text Available Taxonomic assignment of sequence reads is a challenging task in metagenomic data analysis, for which the present methods mainly use either composition- or homology-based approaches. Though the homology-based methods are more sensitive and accurate, they suffer primarily due to the time needed to generate the Blast alignments. We developed the MetaBin program and web server for better homology-based taxonomic assignments using an ORF-based approach. By implementing Blat as the faster alignment method in place of Blastx, the analysis time has been reduced by severalfold. It is benchmarked using both simulated and real metagenomic datasets, and can be used for both single and paired-end sequence reads of varying lengths (≥45 bp. To our knowledge, MetaBin is the only available program that can be used for the taxonomic binning of short reads (<100 bp with high accuracy and high sensitivity using a homology-based approach. The MetaBin web server can be used to carry out the taxonomic analysis, by either submitting reads or Blastx output. It provides several options including construction of taxonomic trees, creation of a composition chart, functional analysis using COGs, and comparative analysis of multiple metagenomic datasets. MetaBin web server and a standalone version for high-throughput analysis are available freely at http://metabin.riken.jp/.

Catalytically Active Guanylyl Cyclase B Requires Endoplasmic Reticulum-mediated Glycosylation, and Mutations That Inhibit This Process Cause Dwarfism.

Science.gov (United States)

Dickey, Deborah M; Edmund, Aaron B; Otto, Neil M; Chaffee, Thomas S; Robinson, Jerid W; Potter, Lincoln R

2016-05-20

C-type natriuretic peptide activation of guanylyl cyclase B (GC-B), also known as natriuretic peptide receptor B or NPR2, stimulates long bone growth, and missense mutations in GC-B cause dwarfism. Four such mutants (L658F, Y708C, R776W, and G959A) bound (125)I-C-type natriuretic peptide on the surface of cells but failed to synthesize cGMP in membrane GC assays. Immunofluorescence microscopy also indicated that the mutant receptors were on the cell surface. All mutant proteins were dephosphorylated and incompletely glycosylated, but dephosphorylation did not explain the inactivation because the mutations inactivated a "constitutively phosphorylated" enzyme. Tunicamycin inhibition of glycosylation in the endoplasmic reticulum or mutation of the Asn-24 glycosylation site decreased GC activity, but neither inhibition of glycosylation in the Golgi by N-acetylglucosaminyltransferase I gene inactivation nor PNGase F deglycosylation of fully processed GC-B reduced GC activity. We conclude that endoplasmic reticulum-mediated glycosylation is required for the formation of an active catalytic, but not ligand-binding domain, and that mutations that inhibit this process cause dwarfism. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Glycosylation status of vitamin D binding protein in cancer patients.

Science.gov (United States)

Rehder, Douglas S; Nelson, Randall W; Borges, Chad R

2009-10-01

On the basis of the results of activity studies, previous reports have suggested that vitamin D binding protein (DBP) is significantly or even completely deglycosylated in cancer patients, eliminating the molecular precursor of the immunologically important Gc macrophage activating factor (GcMAF), a glycosidase-derived product of DBP. The purpose of this investigation was to directly determine the relative degree of O-linked trisaccharide glycosylation of serum-derived DBP in human breast, colorectal, pancreatic, and prostate cancer patients. Results obtained by electrospray ionization-based mass spectrometric immunoassay showed that there was no significant depletion of DBP trisaccharide glycosylation in the 56 cancer patients examined relative to healthy controls. These results suggest that alternative hypotheses regarding the molecular and/or structural origins of GcMAF must be considered to explain the relative inability of cancer patient serum to activate macrophages.

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

Science.gov (United States)

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

2014-01-01

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

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

Directory of Open Access Journals (Sweden)

Robert J Boissy

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

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

Science.gov (United States)

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

A Markov chain model for N-linked protein glycosylation – towards a low-parameter tool for model-driven glycoengineering

DEFF Research Database (Denmark)

Spahn, Philipp N.; Hansen, Anders Holmgaard; Hansen, Henning Gram

2016-01-01

Glycosylation is a critical quality attribute of most recombinant biotherapeutics. Consequently, drug development requires careful control of glycoforms to meet bioactivity and biosafety requirements. However, glycoengineering can be extraordinarily difficult given the complex reaction networks...... present a novel low-parameter approach to describe glycosylation using flux-balance and Markov chain modeling. The model recapitulates the biological complexity of glycosylation, but does not require user-provided kinetic information. We use this method to predict and experimentally validate glycoprofiles...

Insights into resistome and stress responses genes in Bubalus bubalis rumen through metagenomic analysis.

Science.gov (United States)

Reddy, Bhaskar; Singh, Krishna M; Patel, Amrutlal K; Antony, Ancy; Panchasara, Harshad J; Joshi, Chaitanya G

2014-10-01

Buffalo rumen microbiota experience variety of diets and represents a huge reservoir of mobilome, resistome and stress responses. However, knowledge of metagenomic responses to such conditions is still rudimentary. We analyzed the metagenomes of buffalo rumen in the liquid and solid phase of the rumen biomaterial from river buffalo adapted to varying proportion of concentrate to green or dry roughages, using high-throughput sequencing to know the occurrence of antibiotics resistance genes, genetic exchange between bacterial population and environmental reservoirs. A total of 3914.94 MB data were generated from all three treatments group. The data were analysed with Metagenome rapid annotation system tools. At phyla level, Bacteroidetes were dominant in all the treatments followed by Firmicutes. Genes coding for functional responses to stress (oxidative stress and heat shock proteins) and resistome genes (resistance to antibiotics and toxic compounds, phages, transposable elements and pathogenicity islands) were prevalent in similar proportion in liquid and solid fraction of rumen metagenomes. The fluoroquinolone resistance, MDR efflux pumps and Methicillin resistance genes were broadly distributed across 11, 9, and 14 bacterial classes, respectively. Bacteria responsible for phages replication and prophages and phage packaging and rlt-like streptococcal phage genes were mostly assigned to phyla Bacteroides, Firmicutes and proteaobacteria. Also, more reads matching the sigma B genes were identified in the buffalo rumen. This study underscores the presence of diverse mechanisms of adaptation to different diet, antibiotics and other stresses in buffalo rumen, reflecting the proportional representation of major bacterial groups.

Beyond research: a primer for considerations on using viral metagenomics in the field and clinic

OpenAIRE

Hall, Richard J.; Draper, Jenny L.; Nielsen, Fiona G. G.; Dutilh, Bas E.

2015-01-01

Powered by recent advances in next-generation sequencing technologies, metagenomics has already unveiled vast microbial biodiversity in a range of environments, and is increasingly being applied in clinics for difficult-to-diagnose cases. It can be tempting to suggest that metagenomics could be used as a “universal test” for all pathogens without the need to conduct lengthy serial testing using specific assays. While this is an exciting prospect, there are issues that need to be addressed bef...

Marine metagenomics: strategies for the discovery of novel enzymes with biotechnological applications from marine environments

Directory of Open Access Journals (Sweden)

Dobson Alan DW

2008-08-01

Full Text Available Abstract Metagenomic based strategies have previously been successfully employed as powerful tools to isolate and identify enzymes with novel biocatalytic activities from the unculturable component of microbial communities from various terrestrial environmental niches. Both sequence based and function based screening approaches have been employed to identify genes encoding novel biocatalytic activities and metabolic pathways from metagenomic libraries. While much of the focus to date has centred on terrestrial based microbial ecosystems, it is clear that the marine environment has enormous microbial biodiversity that remains largely unstudied. Marine microbes are both extremely abundant and diverse; the environments they occupy likewise consist of very diverse niches. As culture-dependent methods have thus far resulted in the isolation of only a tiny percentage of the marine microbiota the application of metagenomic strategies holds great potential to study and exploit the enormous microbial biodiversity which is present within these marine environments.