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Search Results: 1 - 10 of 9289 matches for " Chongle Pan "
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Measuring Dissociation Rate Constants of Protein Complexes through Subunit Exchange: Experimental Design and Theoretical Modeling
Chongle Pan
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0028827
Abstract: Protein complexes are dynamic macromolecules that constantly dissociate into, and simultaneously are assembled from, free subunits. Dissociation rate constants, koff, provide structural and functional information on protein complexes. However, because all existing methods for measuring koff require high-quality purification and specific modifications of protein complexes, dissociation kinetics has only been studied for a small set of model complexes. Here, we propose a new method, called Metabolically-labeled Affinity-tagged Subunit Exchange (MASE), to measure koff using metabolic stable isotope labeling, affinity purification and mass spectrometry. MASE is based on a subunit exchange process between an unlabeled affinity-tagged variant and a metabolically-labeled untagged variant of a complex. The subunit exchange process was modeled theoretically for a heterodimeric complex. The results showed that koff determines, and hence can be estimated from, the observed rate of subunit exchange. This study provided the theoretical foundation for future experiments that can validate and apply the MASE method.
Phenotype Fingerprinting Suggests the Involvement of Single-Genotype Consortia in Degradation of Aromatic Compounds by Rhodopseudomonas palustris
Tatiana V. Karpinets, Dale A. Pelletier, Chongle Pan, Edward C. Uberbacher, Galina V. Melnichenko, Robert L. Hettich, Nagiza F. Samatova
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0004615
Abstract: Anaerobic degradation of complex organic compounds by microorganisms is crucial for development of innovative biotechnologies for bioethanol production and for efficient degradation of environmental pollutants. In natural environments, the degradation is usually accomplished by syntrophic consortia comprised of different bacterial species. This strategy allows consortium organisms to reduce efforts required for maintenance of the redox homeostasis at each syntrophic level. Cellular mechanisms that maintain the redox homeostasis during the degradation of aromatic compounds by one organism are not fully understood. Here we present a hypothesis that the metabolically versatile phototrophic bacterium Rhodopseudomonas palustris forms its own syntrophic consortia, when it grows anaerobically on p-coumarate or benzoate as a sole carbon source. We have revealed the consortia from large-scale measurements of mRNA and protein expressions under p-coumarate, benzoate and succinate degrading conditions using a novel computational approach referred as phenotype fingerprinting. In this approach, marker genes for known R. palustris phenotypes are employed to determine the relative expression levels of genes and proteins in aromatics versus non-aromatics degrading condition. Subpopulations of the consortia are inferred from the expression of phenotypes and known metabolic modes of the R. palustris growth. We find that p-coumarate degrading conditions may lead to at least three R. palustris subpopulations utilizing p-coumarate, benzoate, and CO2 and H2. Benzoate degrading conditions may also produce at least three subpopulations utilizing benzoate, CO2 and H2, and N2 and formate. Communication among syntrophs and inter-syntrophic dynamics in each consortium are indicated by up-regulation of transporters and genes involved in the curli formation and chemotaxis. The N2-fixing subpopulation in the benzoate degrading consortium has preferential activation of the vanadium nitrogenase over the molybdenum nitrogenase. This subpopulation in the consortium was confirmed in an independent experiment by consumption of dissolved nitrogen gas under the benzoate degrading conditions.
Impact of Pretreated Switchgrass and Biomass Carbohydrates on Clostridium thermocellum ATCC 27405 Cellulosome Composition: A Quantitative Proteomic Analysis
Babu Raman, Chongle Pan, Gregory B. Hurst, Miguel Rodriguez, Catherine K. McKeown, Patricia K. Lankford, Nagiza F. Samatova, Jonathan R. Mielenz
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0005271
Abstract: Background Economic feasibility and sustainability of lignocellulosic ethanol production requires the development of robust microorganisms that can efficiently degrade and convert plant biomass to ethanol. The anaerobic thermophilic bacterium Clostridium thermocellum is a candidate microorganism as it is capable of hydrolyzing cellulose and fermenting the hydrolysis products to ethanol and other metabolites. C. thermocellum achieves efficient cellulose hydrolysis using multiprotein extracellular enzymatic complexes, termed cellulosomes. Methodology/Principal Findings In this study, we used quantitative proteomics (multidimensional LC-MS/MS and 15N-metabolic labeling) to measure relative changes in levels of cellulosomal subunit proteins (per CipA scaffoldin basis) when C. thermocellum ATCC 27405 was grown on a variety of carbon sources [dilute-acid pretreated switchgrass, cellobiose, amorphous cellulose, crystalline cellulose (Avicel) and combinations of crystalline cellulose with pectin or xylan or both]. Cellulosome samples isolated from cultures grown on these carbon sources were compared to 15N labeled cellulosome samples isolated from crystalline cellulose-grown cultures. In total from all samples, proteomic analysis identified 59 dockerin- and 8 cohesin-module containing components, including 16 previously undetected cellulosomal subunits. Many cellulosomal components showed differential protein abundance in the presence of non-cellulose substrates in the growth medium. Cellulosome samples from amorphous cellulose, cellobiose and pretreated switchgrass-grown cultures displayed the most distinct differences in composition as compared to cellulosome samples from crystalline cellulose-grown cultures. While Glycoside Hydrolase Family 9 enzymes showed increased levels in the presence of crystalline cellulose, and pretreated switchgrass, in particular, GH5 enzymes showed increased levels in response to the presence of cellulose in general, amorphous or crystalline. Conclusions/Significance Overall, the quantitative results suggest a coordinated substrate-specific regulation of cellulosomal subunit composition in C. thermocellum to better suit the organism's needs for growth under different conditions. To date, this study provides the most comprehensive comparison of cellulosomal compositional changes in C. thermocellum in response to different carbon sources. Such studies are vital to engineering a strain that is best suited to grow on specific substrates of interest and provide the building blocks for constructing designer cellulosomes with tailored
Phage-Induced Expression of CRISPR-Associated Proteins Is Revealed by Shotgun Proteomics in Streptococcus thermophilus
Jacque C. Young, Brian D. Dill, Chongle Pan, Robert L. Hettich, Jillian F. Banfield, Manesh Shah, Christophe Fremaux, Philippe Horvath, Rodolphe Barrangou, Nathan C. VerBerkmoes
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0038077
Abstract: The CRISPR/Cas system, comprised of clustered regularly interspaced short palindromic repeats along with their associated (Cas) proteins, protects bacteria and archaea from viral predation and invading nucleic acids. While the mechanism of action for this acquired immunity is currently under investigation, the response of Cas protein expression to phage infection has yet to be elucidated. In this study, we employed shotgun proteomics to measure the global proteome expression in a model system for studying the CRISPR/Cas response in S. thermophilus DGCC7710 infected with phage 2972. Host and viral proteins were simultaneously measured following inoculation at two different multiplicities of infection and across various time points using two-dimensional liquid chromatography tandem mass spectrometry. Thirty-seven out of forty predicted viral proteins were detected, including all proteins of the structural virome and viral effector proteins. In total, 1,013 of 2,079 predicted S. thermophilus proteins were detected, facilitating the monitoring of host protein synthesis changes in response to virus infection. Importantly, Cas proteins from all four CRISPR loci in the S. thermophilus DGCC7710 genome were detected, including loci previously thought to be inactive. Many Cas proteins were found to be constitutively expressed, but several demonstrated increased abundance following infection, including the signature Cas9 proteins from the CRISPR1 and CRISPR3 loci, which are key players in the interference phase of the CRISPR/Cas response. Altogether, these results provide novel insights into the proteomic response of S. thermophilus, specifically CRISPR-associated proteins, upon phage 2972 infection.
A high-throughput de novo sequencing approach for shotgun proteomics using high-resolution tandem mass spectrometry
Chongle Pan, Byung H Park, William H McDonald, Patricia A Carey, Jillian F Banfield, Nathan C VerBerkmoes, Robert L Hettich, Nagiza F Samatova
BMC Bioinformatics , 2010, DOI: 10.1186/1471-2105-11-118
Abstract: In this study, a new de novo sequencing algorithm, called Vonode, has been developed specifically for analysis of such high-resolution tandem mass spectra. To fully exploit the high mass accuracy of these spectra, a unique scoring system is proposed to evaluate sequence tags based primarily on mass accuracy information of fragment ions. Consensus sequence tags were inferred for 11,422 spectra with an average peptide length of 5.5 residues from a total of 40,297 input spectra acquired in a 24-hour proteomics measurement of Rhodopseudomonas palustris. The accuracy of inferred consensus sequence tags was 84%. According to our comparison, the performance of Vonode was shown to be superior to the PepNovo v2.0 algorithm, in terms of the number of de novo sequenced spectra and the sequencing accuracy.Here, we improved de novo sequencing performance by developing a new algorithm specifically for high-resolution tandem mass spectral data. The Vonode algorithm is freely available for download at http://compbio.ornl.gov/Vonode webcite.Tandem mass spectrometry (MS/MS) has become an important method for characterizing complex protein mixtures. Since the emergence of this high-throughput technology, two complementary data analysis approaches have been pursued: a database searching approach and a de novo sequencing approach. The former identifies peptide sequences from a protein database by matching their predicted tandem mass spectra to measured tandem mass spectra; whereas the latter infers partial or complete peptide sequences directly from measured tandem mass spectra. Due to its high identification accuracy and the rapid expansion of genomic sequence data, the database searching approach, enabled by popular algorithms such as Sequest [1] and Mascot [2], is routinely used in current proteomics workflows. In contrast, although many de novo algorithms have been developed [3-14], they have not been widely used in high-throughput proteomics workflows. A high-throughput de novo seq
Integrated Metagenomics/Metaproteomics Reveals Human Host-Microbiota Signatures of Crohn's Disease
Alison R. Erickson, Brandi L. Cantarel, Regina Lamendella, Youssef Darzi, Emmanuel F. Mongodin, Chongle Pan, Manesh Shah, Jonas Halfvarson, Curt Tysk, Bernard Henrissat, Jeroen Raes, Nathan C. Verberkmoes, Claire M. Fraser, Robert L. Hettich, Janet K. Jansson
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0049138
Abstract: Crohn's disease (CD) is an inflammatory bowel disease of complex etiology, although dysbiosis of the gut microbiota has been implicated in chronic immune-mediated inflammation associated with CD. Here we combined shotgun metagenomic and metaproteomic approaches to identify potential functional signatures of CD in stool samples from six twin pairs that were either healthy, or that had CD in the ileum (ICD) or colon (CCD). Integration of these omics approaches revealed several genes, proteins, and pathways that primarily differentiated ICD from healthy subjects, including depletion of many proteins in ICD. In addition, the ICD phenotype was associated with alterations in bacterial carbohydrate metabolism, bacterial-host interactions, as well as human host-secreted enzymes. This eco-systems biology approach underscores the link between the gut microbiota and functional alterations in the pathophysiology of Crohn's disease and aids in identification of novel diagnostic targets and disease specific biomarkers.
Strategies for Metagenomic-Guided Whole-Community Proteomics of Complex Microbial Environments
Brandi L. Cantarel, Alison R. Erickson, Nathan C. VerBerkmoes, Brian K. Erickson, Patricia A. Carey, Chongle Pan, Manesh Shah, Emmanuel F. Mongodin, Janet K. Jansson, Claire M. Fraser-Liggett, Robert L. Hettich
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0027173
Abstract: Accurate protein identification in large-scale proteomics experiments relies upon a detailed, accurate protein catalogue, which is derived from predictions of open reading frames based on genome sequence data. Integration of mass spectrometry-based proteomics data with computational proteome predictions from environmental metagenomic sequences has been challenging because of the variable overlap between proteomic datasets and corresponding short-read nucleotide sequence data. In this study, we have benchmarked several strategies for increasing microbial peptide spectral matching in metaproteomic datasets using protein predictions generated from matched metagenomic sequences from the same human fecal samples. Additionally, we investigated the impact of mass spectrometry-based filters (high mass accuracy, delta correlation), and de novo peptide sequencing on the number and robustness of peptide-spectrum assignments in these complex datasets. In summary, we find that high mass accuracy peptide measurements searched against non-assembled reads from DNA sequencing of the same samples significantly increased identifiable proteins without sacrificing accuracy.
Effects of Diet on Resource Utilization by a Model Human Gut Microbiota Containing Bacteroides cellulosilyticus WH2, a Symbiont with an Extensive Glycobiome
Nathan P. McNulty,Meng Wu,Alison R. Erickson,Chongle Pan,Brian K. Erickson,Eric C. Martens,Nicholas A. Pudlo,Brian D. Muegge,Bernard Henrissat,Robert L. Hettich,Jeffrey I. Gordon
PLOS Biology , 2013, DOI: 10.1371/journal.pbio.1001637
Abstract: The human gut microbiota is an important metabolic organ, yet little is known about how its individual species interact, establish dominant positions, and respond to changes in environmental factors such as diet. In this study, gnotobiotic mice were colonized with an artificial microbiota comprising 12 sequenced human gut bacterial species and fed oscillating diets of disparate composition. Rapid, reproducible, and reversible changes in the structure of this assemblage were observed. Time-series microbial RNA-Seq analyses revealed staggered functional responses to diet shifts throughout the assemblage that were heavily focused on carbohydrate and amino acid metabolism. High-resolution shotgun metaproteomics confirmed many of these responses at a protein level. One member, Bacteroides cellulosilyticus WH2, proved exceptionally fit regardless of diet. Its genome encoded more carbohydrate active enzymes than any previously sequenced member of the Bacteroidetes. Transcriptional profiling indicated that B. cellulosilyticus WH2 is an adaptive forager that tailors its versatile carbohydrate utilization strategy to available dietary polysaccharides, with a strong emphasis on plant-derived xylans abundant in dietary staples like cereal grains. Two highly expressed, diet-specific polysaccharide utilization loci (PULs) in B. cellulosilyticus WH2 were identified, one with characteristics of xylan utilization systems. Introduction of a B. cellulosilyticus WH2 library comprising >90,000 isogenic transposon mutants into gnotobiotic mice, along with the other artificial community members, confirmed that these loci represent critical diet-specific fitness determinants. Carbohydrates that trigger dramatic increases in expression of these two loci and many of the organism's 111 other predicted PULs were identified by RNA-Seq during in vitro growth on 31 distinct carbohydrate substrates, allowing us to better interpret in vivo RNA-Seq and proteomics data. These results offer insight into how gut microbes adapt to dietary perturbations at both a community level and from the perspective of a well-adapted symbiont with exceptional saccharolytic capabilities, and illustrate the value of artificial communities.
Systems Biology Analysis of Zymomonas mobilis ZM4 Ethanol Stress Responses
Shihui Yang, Chongle Pan, Timothy J. Tschaplinski, Gregory B. Hurst, Nancy L. Engle, Wen Zhou, PhuongAn Dam, Ying Xu, Miguel Rodriguez, Lezlee Dice, Courtney M. Johnson, Brian H. Davison, Steven D. Brown
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0068886
Abstract: Background Zymomonas mobilis ZM4 is a capable ethanologenic bacterium with high ethanol productivity and ethanol tolerance. Previous studies indicated that several stress-related proteins and changes in the ZM4 membrane lipid composition may contribute to ethanol tolerance. However, the molecular mechanisms of its ethanol stress response have not been elucidated fully. Methodology/Principal Findings In this study, ethanol stress responses were investigated using systems biology approaches. Medium supplementation with an initial 47 g/L (6% v/v) ethanol reduced Z. mobilis ZM4 glucose consumption, growth rate and ethanol productivity compared to that of untreated controls. A proteomic analysis of early exponential growth identified about one thousand proteins, or approximately 55% of the predicted ZM4 proteome. Proteins related to metabolism and stress response such as chaperones and key regulators were more abundant in the early ethanol stress condition. Transcriptomic studies indicated that the response of ZM4 to ethanol is dynamic, complex and involves many genes from all the different functional categories. Most down-regulated genes were related to translation and ribosome biogenesis, while the ethanol-upregulated genes were mostly related to cellular processes and metabolism. Transcriptomic data were used to update Z. mobilis ZM4 operon models. Furthermore, correlations among the transcriptomic, proteomic and metabolic data were examined. Among significantly expressed genes or proteins, we observe higher correlation coefficients when fold-change values are higher. Conclusions Our study has provided insights into the responses of Z. mobilis to ethanol stress through an integrated “omics” approach for the first time. This systems biology study elucidated key Z. mobilis ZM4 metabolites, genes and proteins that form the foundation of its distinctive physiology and its multifaceted response to ethanol stress.
A Contextual Item-Based Collaborative Filtering Technology  [PDF]
Xueqing Tan, Pan Pan
Intelligent Information Management (IIM) , 2012, DOI: 10.4236/iim.2012.43013
Abstract: This paper proposes a contextual item-based collaborative filtering technology, which is based on the traditional item-based collaborative filtering technology. In the process of the recommendation, user’s important mobile contextual information are taken into account, and the technology combines with those ratings on the items in the users’ historical contextual information who are familiar with user’s current context information in order to predict that which items will be preferred by user in his or her current context. At the end, an experiment is used to prove that the technology proposed in this paper can predict user’s preference in his or her mobile environment more accurately.
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