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Find video protocols related to scientific articles indexed in Pubmed.
Flexible and Accessible Workflows for Improved Proteogenomic Analysis Using the Galaxy Framework.
J. Proteome Res.
PUBLISHED: 10-11-2014
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Proteogenomics combines large-scale genomic and transcriptomic data with mass-spectrometry-based proteomic data to discover novel protein sequence variants and improve genome annotation. In contrast with conventional proteomic applications, proteogenomic analysis requires a number of additional data processing steps. Ideally, these required steps would be integrated and automated via a single software platform offering accessibility for wet-bench researchers as well as flexibility for user-specific customization and integration of new software tools as they emerge. Toward this end, we have extended the Galaxy bioinformatics framework to facilitate proteogenomic analysis. Using analysis of whole human saliva as an example, we demonstrate Galaxy's flexibility through the creation of a modular workflow incorporating both established and customized software tools that improve depth and quality of proteogenomic results. Our customized Galaxy-based software includes automated, batch-mode BLASTP searching and a Peptide Sequence Match Evaluator tool, both useful for evaluating the veracity of putative novel peptide identifications. Our complex workflow (approximately 140 steps) can be easily shared using built-in Galaxy functions, enabling their use and customization by others. Our results provide a blueprint for the establishment of the Galaxy framework as an ideal solution for the emerging field of proteogenomics.
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Up-regulation of the Sirtuin 1 (Sirt1) and Peroxisome Proliferator-activated Receptor ? Coactivator-1? (PGC-1?) Genes in White Adipose Tissue of Id1 Protein-deficient Mice: IMPLICATIONS IN THE PROTECTION AGAINST DIET AND AGE-INDUCED GLUCOSE INTOLERANCE.
J. Biol. Chem.
PUBLISHED: 09-04-2014
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Id1, a helix-loop-helix (HLH) protein that inhibits the function of basic HLH E protein transcription factors in lymphoid cells, has been implicated in diet- and age-induced obesity by unknown mechanisms. Here we show that Id1-deficient mice are resistant to a high fat diet- and age-induced obesity, as revealed by reduced weight gain and body fat, increased lipid oxidation, attenuated hepatosteatosis, lower levels of lipid droplets in brown adipose tissue, and smaller white adipocytes after a high fat diet feeding or in aged animals. Id1 deficiency improves glucose tolerance, lowers serum insulin levels, and reduces TNF? gene expression in white adipose tissue. Id1 deficiency also increased expression of Sirtuin 1 and peroxisome proliferator-activated receptor ? coactivator 1?, regulators of mitochondrial biogenesis and energy expenditure, in the white adipose tissue. This effect was accompanied by the elevation of several genes encoding proteins involved in oxidative phosphorylation and fatty acid oxidation, such as cytochrome c, medium-chain acyl-CoA dehydrogenase, and adipocyte protein 2. Moreover, the phenotype for Id1 deficiency was similar to that of mice expressing an E protein dominant-positive construct, ET2, suggesting that the balance between Id and E proteins plays a role in regulating lipid metabolism and insulin sensitivity.
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Using Galaxy-P to leverage RNA-Seq for the discovery of novel protein variations.
BMC Genomics
PUBLISHED: 08-22-2014
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Current practice in mass spectrometry (MS)-based proteomics is to identify peptides by comparison of experimental mass spectra with theoretical mass spectra derived from a reference protein database; however, this strategy necessarily fails to detect peptide and protein sequences that are absent from the database. We and others have recently shown that customized proteomic databases derived from RNA-Seq data can be employed for MS-searching to both improve MS analysis and identify novel peptides. While this general strategy constitutes a significant advance for the discovery of novel protein variations, it has not been readily transferable to other laboratories due to the need for many specialized software tools. To address this problem, we have implemented readily accessible, modifiable, and extensible workflows within Galaxy-P, short for Galaxy for Proteomics, a web-based bioinformatic extension of the Galaxy framework for the analysis of multi-omics (e.g. genomics, transcriptomics, proteomics) data.
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Deficiency in adipocyte chemokine receptor CXCR4 exacerbates obesity and compromises thermoregulatory responses of brown adipose tissue in a mouse model of diet-induced obesity.
FASEB J.
PUBLISHED: 07-11-2014
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The chemokine receptor CXCR4 is expressed on adipocytes and macrophages in adipose tissue, but its role in this tissue remains unknown. We evaluated whether deficiency in either adipocyte or myeloid leukocyte CXCR4 affects body weight (BW) and adiposity in a mouse model of high-fat-diet (HFD)-induced obesity. We found that ablation of adipocyte, but not myeloid leukocyte, CXCR4 exacerbated obesity. The HFD-fed adipocyte-specific CXCR4-knockout (AdCXCR4ko) mice, compared to wild-type C57BL/6 control mice, had increased BW (average: 52.0 g vs. 35.5 g), adiposity (average: 49.3 vs. 21.0% of total BW), and inflammatory leukocyte content in white adipose tissue (WAT), despite comparable food intake. As previously reported, HFD feeding increased uncoupling protein 1 (UCP1) expression (fold increase: 3.5) in brown adipose tissue (BAT) of the C57BL/6 control mice. However, no HFD-induced increase in UCP1 expression was observed in the AdCXCR4ko mice, which were cold sensitive. Thus, our study suggests that adipocyte CXCR4 limits development of obesity by preventing excessive inflammatory cell recruitment into WAT and by supporting thermogenic activity of BAT. Since CXCR4 is conserved between mouse and human, the newfound role of CXCR4 in mouse adipose tissue may parallel the role of this chemokine receptor in human adipose tissue.
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Improved intensity-based label-free quantification via proximity-based intensity normalization (PIN).
J. Proteome Res.
PUBLISHED: 02-26-2014
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Researchers are increasingly turning to label-free MS1 intensity-based quantification strategies within HPLC-ESI-MS/MS workflows to reveal biological variation at the molecule level. Unfortunately, HPLC-ESI-MS/MS workflows using these strategies produce results with poor repeatability and reproducibility, primarily due to systematic bias and complex variability. While current global normalization strategies can mitigate systematic bias, they fail when faced with complex variability stemming from transient stochastic events during HPLC-ESI-MS/MS analysis. To address these problems, we developed a novel local normalization method, proximity-based intensity normalization (PIN), based on the analysis of compositional data. We evaluated PIN against common normalization strategies. PIN outperforms them in dramatically reducing variance and in identifying 20% more proteins with statistically significant abundance differences that other strategies missed. Our results show the PIN enables the discovery of statistically significant biological variation that otherwise is falsely reported or missed.
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Effects of Extreme Climate Events on Tea (Camellia sinensis) Functional Quality Validate Indigenous Farmer Knowledge and Sensory Preferences in Tropical China.
PLoS ONE
PUBLISHED: 01-01-2014
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Climate change is impacting agro-ecosystems, crops, and farmer livelihoods in communities worldwide. While it is well understood that more frequent and intense climate events in many areas are resulting in a decline in crop yields, the impact on crop quality is less acknowledged, yet it is critical for food systems that benefit both farmers and consumers through high-quality products. This study examines tea (Camellia sinensis; Theaceae), the world's most widely consumed beverage after water, as a study system to measure effects of seasonal precipitation variability on crop functional quality and associated farmer knowledge, preferences, and livelihoods. Sampling was conducted in a major tea producing area of China during an extreme drought through the onset of the East Asian Monsoon in order to capture effects of extreme climate events that are likely to become more frequent with climate change. Compared to the spring drought, tea growth during the monsoon period was up to 50% higher. Concurrently, concentrations of catechin and methylxanthine secondary metabolites, major compounds that determine tea functional quality, were up to 50% lower during the monsoon while total phenolic concentrations and antioxidant activity increased. The inverse relationship between tea growth and concentrations of individual secondary metabolites suggests a dilution effect of precipitation on tea quality. The decrease in concentrations of tea secondary metabolites was accompanied by reduced farmer preference on the basis of sensory characteristics as well as a decline of up to 50% in household income from tea sales. Farmer surveys indicate a high degree of agreement regarding climate patterns and the effects of precipitation on tea yields and quality. Extrapolating findings from this seasonal study to long-term climate scenario projections suggests that farmers and consumers face variable implications with forecasted precipitation scenarios and calls for research on management practices to facilitate climate adaptation for sustainable crop production.
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A feasibility study to identify proteins in the residual Pap test fluid of women with normal cytology by mass spectrometry-based proteomics.
Clin Proteomics
PUBLISHED: 01-01-2014
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The proteomic analysis of body fluids is a growing technology for the identification of protein biomarkers of disease. Given that Papanicolaou tests (Pap tests) are routinely performed on over 30 million women annually in the U.S. to screen for cervical cancer, we examined the residual Pap test fluid as a source of protein for analysis by mass spectrometry (MS). In the liquid-based Pap test, cervical cells are collected from the ectocervix and placed into an alcohol-based fixative prior to staining and pathologic examination. We hypothesized that proteins shed by cells of the female genital tract can be detected in the Pap test fixative by MS-based proteomic techniques. We examined the feasibility of using residual fluid from discarded Pap tests with cytologically "normal" results to optimize sample preparation for MS analysis. The protein composition of the cell-free Pap test fluid was determined by silver staining of sodium dodecyl sulfate -polyacrylamide gels, and the abundance of serum proteins was examined by Western immunoblot using an antibody against human serum albumin. Both pooled and individual samples were trypsin digested and analyzed by two-dimensional MS/MS. Proteins were identified by searching against the Human Uniprot database, and characterized for localization, function and relative abundance.
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Effects of water availability and pest pressures on tea (Camellia sinensis) growth and functional quality.
AoB Plants
PUBLISHED: 01-01-2014
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Extreme shifts in water availability linked to global climate change are impacting crops worldwide. The present study examines the direct and interactive effects of water availability and pest pressures on tea (Camellia sinensis; Theaceae) growth and functional quality. Manipulative greenhouse experiments were used to measure the effects of variable water availability and pest pressures simulated by jasmonic acid (JA) on tea leaf growth and secondary metabolites that determine tea quality. Water treatments were simulated to replicate ideal tea growing conditions and extreme precipitation events in tropical southwestern China, a major centre of tea production. Results show that higher water availability and JA significantly increased the growth of new leaves while their interactive effect was not significant. The effect of water availability and JA on tea quality varied with individual secondary metabolites. Higher water availability significantly increased total methylxanthine concentrations of tea leaves but there was no significant effect of JA treatments or the interaction of water and JA. Water availability, JA treatments or their interactive effects had no effect on the concentrations of epigallocatechin 3-gallate. In contrast, increased water availability resulted in significantly lower concentrations of epicatechin 3-gallate but the effect of JA and the interactive effects of water and JA were not significant. Lastly, higher water availability resulted in significantly higher total phenolic concentrations but there was no significant impact of JA and their interaction. These findings point to the fascinating dynamics of climate change effects on tea plants with offsetting interactions between precipitation and pest pressures within agro-ecosystems, and the need for future climate studies to examine interactive biotic and abiotic effects.
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Quantitative proteomic analysis of oral brush biopsies identifies secretory leukocyte protease inhibitor as a promising, mechanism-based oral cancer biomarker.
PLoS ONE
PUBLISHED: 01-01-2014
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A decrease in the almost fifty percent mortality rate from oral cancer is needed urgently. Improvements in early diagnosis and more effective preventive treatments could affect such a decrease. Towards this end, we undertook for the first time an in-depth mass spectrometry-based quantitative shotgun proteomics study of non-invasively collected oral brush biopsies. Proteins isolated from brush biopsies from healthy normal tissue, oral premalignant lesion tissue (OPMLs), oral squamous cell carcinoma (OSCC) and matched control tissue were compared. In replicated proteomic datasets, the secretory leukocyte protease inhibitor (SLPI) protein stood out based on its decrease in abundance in both OPML and OSCC lesion tissues compared to healthy normal tissue. Western blotting in additional brushed biopsy samples confirmed a trend of gradual decreasing SLPI abundance between healthy normal and OPML tissue, with a larger decrease in OSCC lesion tissue. A similar SLPI decrease was observed in-vitro comparing model OPML and OSCC cell lines. In addition, exfoliated oral cells in patients' whole saliva showed a loss of SLPI correlated with oral cancer progression. These results, combined with proteomics data indicating a decrease in SLPI in matched healthy control tissue from OSCC patients compared to tissue from healthy normal tissue, suggested a systemic decrease of SLPI in oral cells correlated with oral cancer development. Finally, in-vitro experiments showed that treatment with SLPI significantly decreased NF-kB activity in an OPML cell line. The findings indicate anti-inflammatory activity in OPML, supporting a mechanistic role of SLPI in OSCC progression and suggesting its potential for preventative treatment of at-risk oral lesions. Collectively, our results show for the first time the potential for SLPI as a mechanism-based, non-invasive biomarker of oral cancer progression with potential in preventive treatment.
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Calibration of a semi-automated segmenting method for quantification of adipose tissue compartments from magnetic resonance images of mice.
Metab. Clin. Exp.
PUBLISHED: 03-01-2013
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To use an automated water-suppressed magnetic resonance imaging (MRI) method to objectively assess adipose tissue (AT) volumes in whole body and specific regional body components (subcutaneous, thoracic and peritoneal) of obese and lean mice.
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Mass spectrometry-based proteomics: basic principles and emerging technologies and directions.
Adv. Exp. Med. Biol.
PUBLISHED: 02-05-2013
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As the main catalytic and structural molecules within living systems, proteins are the most likely biomolecules to be affected by radiation exposure. Proteomics, the comprehensive characterization of proteins within complex biological samples, is therefore a research approach ideally suited to assess the effects of radiation exposure on cells and tissues. For comprehensive characterization of proteomes, an analytical platform capable of quantifying protein abundance, identifying post-translation modifications and revealing members of protein complexes on a system-wide level is necessary. Mass spectrometry (MS), coupled with technologies for sample fractionation and automated data analysis, provides such a versatile and powerful platform. In this chapter we offer a view on the current state of MS-proteomics, and focus on emerging technologies within three areas: (1) New instrumental methods; (2) New computational methods for peptide identification; and (3) Label-free quantification. These emerging technologies should be valuable for researchers seeking to better understand biological effects of radiation on living systems.
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A two-step database search method improves sensitivity in peptide sequence matches for metaproteomics and proteogenomics studies.
Proteomics
PUBLISHED: 01-19-2013
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Large databases (>10(6) sequences) used in metaproteomic and proteogenomic studies present challenges in matching peptide sequences to MS/MS data using database-search programs. Most notably, strict filtering to avoid false-positive matches leads to more false negatives, thus constraining the number of peptide matches. To address this challenge, we developed a two-step method wherein matches derived from a primary search against a large database were used to create a smaller subset database. The second search was performed against a target-decoy version of this subset database merged with a host database. High confidence peptide sequence matches were then used to infer protein identities. Applying our two-step method for both metaproteomic and proteogenomic analysis resulted in twice the number of high confidence peptide sequence matches in each case, as compared to the conventional one-step method. The two-step method captured almost all of the same peptides matched by the one-step method, with a majority of the additional matches being false negatives from the one-step method. Furthermore, the two-step method improved results regardless of the database search program used. Our results show that our two-step method maximizes the peptide matching sensitivity for applications requiring large databases, especially valuable for proteogenomics and metaproteomics studies.
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Three-dimensional peptide fractionation for highly sensitive nanoscale LC-based shotgun proteomic analysis of complex protein mixtures.
Methods Mol. Biol.
PUBLISHED: 09-28-2011
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To sensitively analyze complex protein mixtures by mass spectrometry-based shotgun proteomics, researchers have employed platforms that couple orthogonal peptide fractionation methods using nanoscale HPLC. Commonly used platforms have coupled either strong cation exchange (SCX) HPLC or preparative isoelectric focusing (IEF) with nanoscale reversed-phase (nanoRP) HPLC fractionation of peptides. Coupling two dimensions of peptide fractionation, prior to mass spectrometric analysis, increases the sensitivity for identifying low abundance proteins. However, the large dynamic range of protein abundance and high level of complexity of protein mixtures derived from many biological sources, such as bodily fluids, require additional steps of peptide fractionation. To address this shortcoming, we have developed a platform combining three dimensions of peptide fractionation as follows: (1) preparative IEF; (2) SCX HPLC; and (3) nanoRP HPLC. This platform significantly increases the sensitivity of shotgun proteomic analysis in complex protein mixtures. Here, we describe the implementation of this three-dimensional peptide fractionation platform for proteomic studies of complex mixtures.
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Evaluating the potential of a novel oral lesion exudate collection method coupled with mass spectrometry-based proteomics for oral cancer biomarker discovery.
Clin Proteomics
PUBLISHED: 07-27-2011
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Early diagnosis of Oral Squamous Cell Carcinoma (OSCC) increases the survival rate of oral cancer. For early diagnosis, molecular biomarkers contained in samples collected non-invasively and directly from at-risk oral premalignant lesions (OPMLs) would be ideal.
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Large-scale phosphoproteomics analysis of whole saliva reveals a distinct phosphorylation pattern.
J. Proteome Res.
PUBLISHED: 03-01-2011
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In-depth knowledge of bodily fluid phosphoproteomes, such as whole saliva, is limited. To better understand the whole saliva phosphoproteome, we generated a large-scale catalog of phosphorylated proteins. To circumvent the wide dynamic range of phosphoprotein abundance in whole saliva, we combined dynamic range compression using hexapeptide beads, strong cation exchange HPLC peptide fractionation, and immobilized metal affinity chromatography prior to mass spectrometry. In total, 217 unique phosphopeptides sites were identified representing 85 distinct phosphoproteins at 2.3% global FDR. From these peptides, 129 distinct phosphorylation sites were identified of which 57 were previously known, but only 11 of which had been previously identified in whole saliva. Cellular localization analysis revealed salivary phosphoproteins had a distribution similar to all known salivary proteins, but with less relative representation in "extracellular" and "plasma membrane" categories compared to salivary glycoproteins. Sequence alignment showed that phosphorylation occurred at acidic-directed kinase, proline-directed, and basophilic motifs. This differs from plasma phosphoproteins, which predominantly occur at Golgi casein kinase recognized sequences. Collectively, these results suggest diverse functions for salivary phosphoproteins and multiple kinases involved in their processing and secretion. In all, this study should lay groundwork for future elucidation of the functions of salivary protein phosphorylation.
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Effects of different potato cropping system approaches and water management on soilborne diseases and soil microbial communities.
Phytopathology
PUBLISHED: 02-25-2011
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Four different potato cropping systems, designed to address specific management goals of soil conservation, soil improvement, disease suppression, and a status quo standard rotation control, were evaluated for their effects on soilborne diseases of potato and soil microbial community characteristics. The status quo system (SQ) consisted of barley underseeded with red clover followed by potato (2-year). The soil-conserving system (SC) featured an additional year of forage grass and reduced tillage (3-year, barley/timothy-timothy-potato). The soil-improving system (SI) added yearly compost amendments to the SC rotation, and the disease-suppressive system (DS) featured diverse crops with known disease-suppressive capability (3-year, mustard/rapeseed-sudangrass/rye-potato). Each system was also compared with a continuous potato control (PP) and evaluated under both irrigated and nonirrigated conditions. Data collected over three potato seasons following full rotation cycles demonstrated that all rotations reduced stem canker (10 to 50%) relative to PP. The SQ, SC, and DS systems reduced black scurf (18 to 58%) relative to PP; SI reduced scurf under nonirrigated but not irrigated conditions; and scurf was lower in DS than all other systems. The SQ, SC, and DS systems also reduced common scab (15 to 45%), and scab was lower in DS than all other systems. Irrigation increased black scurf and common scab but also resulted in higher yields for most rotations. SI produced the highest yields under nonirrigated conditions, and DS produced high yields and low disease under both irrigation regimes. Each cropping system resulted in distinctive changes in soil microbial community characteristics as represented by microbial populations, substrate utilization, and fatty acid methyl-ester (FAME) profiles. SI tended to increase soil moisture, microbial populations, and activity, as well result in higher proportions of monounsaturated FAMEs and the FAME biomarker for mycorrhizae (16:1 ?6c) relative to most other rotations. DS resulted in moderate microbial populations and activity but higher substrate richness and diversity in substrate utilization profiles. DS also resulted in relatively higher proportions of FAME biomarkers for fungi (18:2 ?6c), actinomycetes, and gram-positive bacteria than most other systems, whereas PP resulted in the lowest microbial populations and activity; substrate richness and diversity; proportions of monounsaturated and polyunsaturated FAME classes; and fungal, mycorrhizae, and actinomycete FAME biomarkers of all cropping systems. Overall, soil water, soil quality, and soilborne diseases were all important factors affecting productivity, and cropping systems addressing these constraints improved production. Cropping system approaches will need to balance these factors to achieve sustainable production and disease management.
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Sample collection and handling considerations for peptidomic studies in whole saliva; implications for biomarker discovery.
Clin. Chim. Acta
PUBLISHED: 01-20-2011
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Proteomic studies in saliva have demonstrated its potential as a diagnostic biofluid, however the salivary peptidome is less studied. Here we study the effects of several sample collection and handling factors on salivary peptide abundance levels.
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Hexapeptide libraries for enhanced protein PTM identification and relative abundance profiling in whole human saliva.
J. Proteome Res.
PUBLISHED: 01-14-2011
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Dynamic range compression (DRC) by hexapeptide libraries increases MS/MS-based identification of lower-abundance proteins in complex mixtures. However, two unanswered questions impede fully realizing DRCs potential in shotgun proteomics. First, does DRC enhance identification of post-translationally modified proteins? Second, can DRC be incorporated into a workflow enabling relative protein abundance profiling? We sought to answer both questions analyzing human whole saliva. Addressing question one, we coupled DRC with covalent glycopeptide enrichment and MS/MS. With DRC we identified ?2 times more N-linked glycoproteins and their glycosylation sites than without DRC, dramatically increasing the known salivary glycoprotein catalog. Addressing question two, we compared differentially stable isotope-labeled saliva samples pooled from healthy and metastatic breast cancer women using a multidimensional peptide fractionation-based workflow, analyzing in parallel one sample portion with DRC and one portion without. Our workflow categorizes proteins with higher absolute abundance, whose relative abundance ratios are altered by DRC, from proteins of lower absolute abundance detected only after DRC. Within each of these salivary protein categories, we identified novel abundance changes putatively associated with breast cancer, demonstrating feasibility and benefits of DRC for relative abundance profiling. Collectively, our results bring us closer to realizing the full potential of DRC for proteomic studies.
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Mass spectrometric identification of phosphorylation sites in guanylyl cyclase A and B.
Biochemistry
PUBLISHED: 11-08-2010
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Guanylyl cyclase A and B (GC-A and GC-B) are transmembrane guanylyl cyclase receptors that mediate the physiologic effects of natriuretic peptides. Some sites of phosphorylation are known for rat GC-A and GC-B, but no phosphorylation site information is available for the human homologues. Here, we used mass spectrometry to identify phosphorylation sites in GC-A and GC-B from both species. Tryptic digests of receptors purified from HEK293 cells were separated and analyzed by nLC-MS-MS. Seven sites of phosphorylation were identified in rat GC-A (S497, T500, S502, S506, S510, T513, and S487), and all of these sites except S510 and T513 were observed in human GC-A. Six phosphorylation sites were identified in rat GC-B (S513, T516, S518, S523, S526, and T529), and all six sites were also identified in human GC-B. Five sites are identical between GC-A and GC-B. S487 in GC-A and T529 in GC-B are novel, uncharacterized sites. Substitution of alanine for S487 did not affect initial ligand-dependent GC-A activity, but a glutamate substitution reduced activity 20%. Similar levels of ANP-dependent desensitization were observed for the wild-type, S487A, and S487E forms of GC-A. Substitution of glutamate or alanine for T529 increased or decreased ligand-dependent cyclase activity of GC-B, respectively, and T529E increased cyclase activity in a GC-B mutant containing glutamates for all five previously identified sites as well. In conclusion, we identified and characterized new phosphorylation sites in GC-A and GC-B and provide the first evidence of phosphorylation sites within human guanylyl cyclases.
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LTQ-iQuant: A freely available software pipeline for automated and accurate protein quantification of isobaric tagged peptide data from LTQ instruments.
Proteomics
PUBLISHED: 09-08-2010
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Pulsed Q dissociation enables combining LTQ ion trap instruments with isobaric peptide tagging. Unfortunately, this combination lacks a technique which accurately reports protein abundance ratios and is implemented in a freely available, flexible software pipeline. We developed and implemented a technique assigning collective reporter ion intensity-based weights to each peptide abundance ratio and calculating a proteins weighted average abundance ratio and p-value. Using an iTRAQ-labeled standard mixture, we compared our techniques performance to the commercial software MASCOT, finding that it performed better than MASCOTs nonweighted averaging and median peptide ratio techniques, and equal to its weighted averaging technique. We also compared performance of the LTQ-Orbitrap plus our technique to 4800 MALDI TOF/TOF plus Protein Pilot, by analyzing an iTRAQ-labeled stem cell lysate. We found highly correlated protein abundance ratios, indicating that the LTQ-Orbitrap plus our technique yields results comparable to the current standard. We implemented our technique in a freely available, automated software pipeline, called LTQ-iQuant, which is mzXML-compatible; supports iTRAQ 4-plex and 8-plex LTQ data; and can be modified for and have weights trained to a users LTQ and other isobaric peptide tagging methods. LTQ-iQuant should make LTQ instruments and isobaric peptide tagging accessible to more proteomic researchers.
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Novel In Situ Collection of Tumor Interstitial Fluid from a Head and Neck Squamous Carcinoma Reveals a Unique Proteome with Diagnostic Potential.
Clin Proteomics
PUBLISHED: 07-25-2010
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INTRODUCTION: Tumors lack normal drainage of secreted fluids and consequently build up tumor interstitial fluid (TIF). Unlike other bodily fluids, TIF likely contains a high proportion of tumor-specific proteins with potential as biomarkers. METHODS: Here, we evaluated a novel technique using a unique ultrafiltration catheter for in situ collection of TIF and used it to generate the first catalog of TIF proteins from a head and neck squamous cell carcinoma (HNSCC). To maximize proteomic coverage, TIF was immunodepleted for high abundance proteins and digested with trypsin, and peptides were fractionated in three dimensions prior to mass spectrometry. RESULTS: We identified 525 proteins with high confidence. The HNSCC TIF proteome was distinct compared to proteomes of other bodily fluids. It contained a relatively high proportion of proteins annotated by Gene Ontology as "extracellular" compared to other secreted fluid and cellular proteomes, indicating minimal cell lysis from our in situ collection technique. Several proteins identified are putative biomarkers of HNSCC, supporting our catalogs value as a source of potential biomarkers. CONCLUSIONS: In all, we demonstrate a reliable new technique for in situ TIF collection and provide the first HNSCC TIF protein catalog with value as a guide for others seeking to develop tumor biomarkers. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12014-010-9050-3) contains supplementary material, which is available to authorized users.
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Safe haven laws as crime control theater.
Child Abuse Negl
PUBLISHED: 06-10-2010
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This article examines safe haven laws, which allow parents to legally abandon their infants. The main objective is to determine whether safe haven laws fit the criteria of crime control theater, a term used to describe public policies that produce the appearance, but not the effect, of crime control, and as such are essentially socially constructed "solutions" to socially constructed crime "problems."
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Quantitative proteomics reveals myosin and actin as promising saliva biomarkers for distinguishing pre-malignant and malignant oral lesions.
PLoS ONE
PUBLISHED: 04-12-2010
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Oral cancer survival rates increase significantly when it is detected and treated early. Unfortunately, clinicians now lack tests which easily and reliably distinguish pre-malignant oral lesions from those already transitioned to malignancy. A test for proteins, ones found in non-invasively-collected whole saliva and whose abundances distinguish these lesion types, would meet this critical need.
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Locomotor activity and gait in aged mice deficient for type IX collagen.
J. Appl. Physiol.
PUBLISHED: 04-01-2010
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Osteoarthritis (OA) is a risk factor for physical inactivity and impaired mobility, but it is not well understood how these locomotor behaviors are affected by the age of onset of OA and disease severity. Male mice homozygous for a Col9a1 gene inactivation (Col9a1(-/-)) develop early onset knee OA, increased tactile pain sensitivity, and gait alterations by 9 mo of age. We hypothesized that aged Col9a1(-/-) mice would reduce joint pain by adopting locomotor behaviors that reduce both the magnitude and daily frequency of joint loading. We tested this hypothesis by evaluating gait and spontaneous locomotor activity in 15- to 17-mo-old male Col9a1(-/-) (n = 5) and Col9a1(+/+)(WT) (n = 5) mice using well-controlled measures of voluntary activity in overground and running wheel conditions, as well as studies of gait in a velocity-controlled treadmill. We found no difference due to genotype in freely chosen locomotor velocity, stride frequency, hindfoot duty factor, dark phase activity time, or dark-phase travel distance during overground, running wheel, or speed-matched treadmill locomotion. Interpretation of these findings is potentially confounded by the observation that WT mice have greater knee OA than Col9a1(-/-) mice in the lateral tibial plateau by 17 mo of age. When accounting for individual differences in knee OA, functional locomotor impairments in aged Col9a1(-/-) and WT mice are manifested as reductions in total locomotor activity levels (e.g., both distance traveled and time active), particularly for wheel running. These results support the concept that current disease status, rather than age of disease onset, is the primary determinant of impaired locomotor activity with aging.
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Diet-induced obesity differentially regulates behavioral, biomechanical, and molecular risk factors for osteoarthritis in mice.
Arthritis Res. Ther.
PUBLISHED: 03-18-2010
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Obesity is a major risk factor for the development of osteoarthritis in both weight-bearing and nonweight-bearing joints. The mechanisms by which obesity influences the structural or symptomatic features of osteoarthritis are not well understood, but may include systemic inflammation associated with increased adiposity. In this study, we examined biomechanical, neurobehavioral, inflammatory, and osteoarthritic changes in C57BL/6J mice fed a high-fat diet.
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Multitagging proteomic strategy to estimate protein turnover rates in dynamic systems.
J. Proteome Res.
PUBLISHED: 02-27-2010
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Current techniques for quantitative proteomics focus mainly on measuring overall protein dynamics, which is the net result of protein synthesis and degradation. Understanding the rate of this synthesis/degradation is essential to fully appreciate cellular dynamics and bridge the gap between transcriptome and proteome data. Protein turnover rates can be estimated through "label-chase" experiments employing stable isotope-labeled precursors; however, the implicit assumption of steady-state in such analyses may not be applicable for many intrinsically dynamic systems. In this study, we present a novel extension of the "label-chase" concept using SILAC and a secondary labeling step with iTRAQ reagents to estimate protein turnover rates in Streptomyces coelicolor cultures undergoing transition from exponential growth to stationary phase. Such processes are of significance in Streptomyces biology as they pertain to the onset of synthesis of numerous therapeutically important secondary metabolites. The dual labeling strategy enabled decoupling of labeled peptide identification and quantification of degradation dynamics at MS and MS/MS scans respectively. Tandem mass spectrometry analysis of these multitagged proteins enabled estimation of degradation rates for 115 highly abundant proteins in S. coelicolor. We compared the rate constants obtained using this dual labeling approach with those from a SILAC-only analysis (assuming steady-state) and show that significant differences are generally observed only among proteins displaying considerable temporal dynamics and that the directions of these differences are largely consistent with theoretical predictions.
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Global assessment of protein turnover in recombinant antibody producing myeloma cells.
J. Biotechnol.
PUBLISHED: 02-03-2010
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The global turnover rates of cellular proteins and the secretion rate of a recombinant immunoglobulin G (IgG) in a myeloma cell line, NS0, were determined using SILAC proteomic analysis. After complete labeling of cellular proteins with (13)C(6), (15)N(4)-arginine, cells were transferred to unlabeled medium and the decay of the labeled arginine in proteins was monitored during exponential cell growth. After PAGE separation and mass-spectrometric identification of proteins, those detected with high confidence over at least three time points were used for the determination of turnover rates. Among the 224 proteins quantified with a protein half-life, about 15% have a degradation rate constant lower than one-tenth of specific growth rate. For most proteins, the turnover rate is insignificant in its overall dynamics. Only 6.3% of proteins have a half-life shorter than the cell doubling time. For IgG secretion, both heavy and light chain molecules follow the same kinetic behavior with a half-life estimated to be 2h. The label decay curve appears to show a second region with very slow kinetics, raising the possibility of two populations of IgG molecules with different secretion characteristics.
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Targeted 18O-labeling for improved proteomic analysis of carbonylated peptides by mass spectrometry.
J. Am. Soc. Mass Spectrom.
PUBLISHED: 02-03-2010
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Proteomic characterization of carbonylated amino acid sites currently relies on confidently matching tandem mass spectra (MS(2)) to peptides within a sequence database. Although effective to some degree, reliable proteomic characterization of carbonylated peptides using this approach remains a challenge needing new, complementary solutions. To this end, we developed a method based on partial (18)O-labeling of reactive carbonyl modifications, which produces a unique isotope signature in mass spectra of carbonylated peptides and enables their detection without reliance on matching MS(2) spectra to a peptide sequence. Key to our method were optimized measures for eliminating trypsin-catalyzed incorporation of (18)O at peptide C-termini, and for stabilizing the incorporated (18)O within the carbonyl modification to prevent its loss during liquid chromatography separation. Applying our method to a rat skeletal muscle homogenate treated with the carbonyl modification 4-hyroxynonenal (4-HNE), we demonstrated its compatibility with solid-phase hydrazide enrichment of carbonylated peptides from complex mixtures. Additionally, we demonstrated the value of (18)O isotope signatures for confirming HNE-modified peptide sequences matched via sequence database searching, and identifying modified peptides missed by MS(2) and/or sequence database searching. Combining our (18)O-labeling method with a customized automated software script, we systematically evaluated for the first time the efficiency of MS(2) and sequence database searching for identifying HNE-modified peptides. We estimated that less than half of the modified peptides selected for MS(2) were successfully identified. Collectively, our method and software should provide valuable new tools for investigators studying protein carbonylation via mass spectrometry-based proteomics.
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Quantitative nuclear proteomics identifies mTOR regulation of DNA damage response.
Mol. Cell Proteomics
PUBLISHED: 11-23-2009
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Cellular nutritional and energy status regulates a wide range of nuclear processes important for cell growth, survival, and metabolic homeostasis. Mammalian target of rapamycin (mTOR) plays a key role in the cellular responses to nutrients. However, the nuclear processes governed by mTOR have not been clearly defined. Using isobaric peptide tagging coupled with linear ion trap mass spectrometry, we performed quantitative proteomics analysis to identify nuclear processes in human cells under control of mTOR. Within 3 h of inhibiting mTOR with rapamycin in HeLa cells, we observed down-regulation of nuclear abundance of many proteins involved in translation and RNA modification. Unexpectedly, mTOR inhibition also down-regulated several proteins functioning in chromosomal integrity and up-regulated those involved in DNA damage responses (DDRs) such as 53BP1. Consistent with these proteomic changes and DDR activation, mTOR inhibition enhanced interaction between 53BP1 and p53 and increased phosphorylation of ataxia telangiectasia mutated (ATM) kinase substrates. ATM substrate phosphorylation was also induced by inhibiting protein synthesis and suppressed by inhibiting proteasomal activity, suggesting that mTOR inhibition reduces steady-state (abundance) levels of proteins that function in cellular pathways of DDR activation. Finally, rapamycin-induced changes led to increased survival after radiation exposure in HeLa cells. These findings reveal a novel functional link between mTOR and DDR pathways in the nucleus potentially operating as a survival mechanism against unfavorable growth conditions.
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Systematic comparison of the human saliva and plasma proteomes.
Proteomics Clin Appl
PUBLISHED: 11-10-2009
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The proteome of human salivary fluid has the potential to open new doors for disease biomarker discovery. A recent study to comprehensively identify and catalog the human ductal salivary proteome led to the compilation of 1166 proteins. The protein complexity of both saliva and plasma is large, suggesting that a comparison of these two proteomes will provide valuable insight into their physiological significance and an understanding of the unique and overlapping disease diagnostic potential that each fluid provides. To create a more comprehensive catalog of human salivary proteins, we have first compiled an extensive list of proteins from whole saliva (WS) identified through MS experiments. The WS list is thereafter combined with the proteins identified from the ductal parotid, and submandibular and sublingual (parotid/SMSL) salivas. In parallel, a core dataset of the human plasma proteome with 3020 protein identifications was recently released. A total of 1939 nonredundant salivary proteins were compiled from a total of 19 474 unique peptide sequences identified from whole and ductal salivas; 740 out of the total 1939 salivary proteins were identified in both whole and ductal saliva. A total of 597 of the salivary proteins have been observed in plasma. Gene ontology (GO) analysis showed similarities in the distributions of the saliva and plasma proteomes with regard to cellular localization, biological processes, and molecular function, but revealed differences which may be related to the different physiological functions of saliva and plasma. The comprehensive catalog of the salivary proteome and its comparison to the plasma proteome provides insights useful for future study, such as exploration of potential biomarkers for disease diagnostics.
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A dynamic range compression and three-dimensional peptide fractionation analysis platform expands proteome coverage and the diagnostic potential of whole saliva.
J. Proteome Res.
PUBLISHED: 10-10-2009
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Comprehensive identification of proteins in whole human saliva is critical for appreciating its full diagnostic potential. However, this is challenged by the large dynamic range of protein abundance within the fluid. To address this problem, we used an analysis platform that coupled hexapeptide libraries for dynamic range compression (DRC) with three-dimensional (3D) peptide fractionation. Our approach identified 2340 proteins in whole saliva and represents the largest saliva proteomic dataset generated using a single analysis platform. Three-dimensional peptide fractionation involving sequential steps of preparative isoelectric focusing (IEF), strong cation exchange, and capillary reversed-phase liquid chromatography was essential for maximizing gains from DRC. Compared to saliva not treated with hexapeptide libraries, DRC substantially increased identified proteins across physicochemical and functional categories. Approximately 20% of total salivary proteins are also seen in plasma, and proteins in both fluids show comparable functional diversity and disease-linkage. However, for a subset of diseases, saliva has higher apparent diagnostic potential. These results expand the potential for whole saliva in health monitoring/diagnostics and provide a general platform for improving proteomic coverage of complex biological samples.
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Extreme obesity due to impaired leptin signaling in mice does not cause knee osteoarthritis.
Arthritis Rheum.
PUBLISHED: 10-01-2009
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To test the hypothesis that obesity resulting from deletion of the leptin gene or the leptin receptor gene results in increased knee osteoarthritis (OA), systemic inflammation, and altered subchondral bone morphology.
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Decreased physical function and increased pain sensitivity in mice deficient for type IX collagen.
Arthritis Rheum.
PUBLISHED: 08-29-2009
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In mice with Col9a1 gene inactivation (Col9a1(-/-)), osteoarthritis (OA) and intervertebral disc degeneration develop prematurely. The aim of this study was to investigate Col9a1(-/-) mice for functional and symptomatic changes that may be associated with these pathologies.
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A study of rituximab and ifosfamide, carboplatin, and etoposide chemotherapy in children with recurrent/refractory B-cell (CD20+) non-Hodgkin lymphoma and mature B-cell acute lymphoblastic leukemia: a report from the Childrens Oncology Group.
Pediatr Blood Cancer
PUBLISHED: 02-07-2009
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To estimate the response rate and therapy related toxicities of the anti-CD20 monoclonal antibody rituximab when combined with chemotherapy including ifosfamide, carboplatin, and etoposide (ICE) in patients with relapsed and refractory B-cell non-Hodgkin lymphoma and mature B-cell acute lymphoblastic leukemia (B-ALL).
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Phosphorus forms in conventional and organic dairy manure identified by solution and solid state p-31 NMR spectroscopy.
J. Environ. Qual.
PUBLISHED: 01-01-2009
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Organic dairy production has increased rapidly in recent years. Organic dairy cows (Bos taurus) generally eat different diets than their conventional counterparts. Although these differences could impact availability, utilization, and cycling of manure nutrients, little such information is available to aid organic dairy farmers in making nutrient and manure management decisions. In this study, we comparatively characterized P in organic and conventional dairy manure using solution and solid state (31)P NMR spectroscopic techniques. Phosphorus in both types of dairy manure was extracted with water, Na acetate buffer (100 mmol L(-1), pH 5.0) plus 20 mg Na dithionite mL(-1), or 0.025 mol L(-1) NaOH with 50 mmolL(-1) EDTA. Solution NMR analysis revealed that organic dairy manure contained about 10% more inorganic phosphate than conventional dairy manure. Whereas organic dairy manure did contain slightly more phytate P, it contained 30 to 50% less monoester P than conventional dairy manure. Solid state NMR spectroscopy revealed that mono-, di-, and trivalent metal P species with different stabilities were present in the two dairy manures. Conventional dairy manure contained relatively higher contents of soluble inorganic P species and stable metal phytate species. In contrast, organic dairy manure contained more Ca and Mg species of P. These results indicate that P transformation rates and quantities should be expected to differ between organic and conventional dairy manures.
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Effectiveness and efficacy of conservation options after potato harvest.
J. Environ. Qual.
PUBLISHED: 01-01-2009
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Soil erosion and phosphorus (P) runoff can be severe in potato production systems in the Northeast USA, which are characterized by intensive tillage, minimal ground cover, low crop residue return, and steep slopes. We used rainfall simulators in the greenhouse and field to assess sediment and P movement associated with two conservation practices: straw mulching and application of polyacrylamide (PAM). In the greenhouse, a Nokomis sandy loam soil (fine-loamy, mixed, frigid Typic Haplorthods) was packed into 0.2 by 1.0 m boxes and subjected to four rainfall events at an intensity of 70 mm h(-1). Runoff amount, sediment concentration, and inorganic and sediment-bound P were measured for 30 min after initiation of runoff. Linear increases in straw mulch biomass (up to equivalent of 3000 kg ha(-1)) resulted in exponential decreases in sediment and P loss. Mulch applied at rates as low as 600 kg ha(-1) provided nearly 50% ground cover and reduced sediment movement and sediment-bound P concentration and loss by >50%. Higher application rates reduced sediment loss by up to 95% but contributed dissolved reactive P (DRP) to runoff water. Field observations using simulated rainfall on mulch-covered and bare soil were consistent with greenhouse results. Linear increases in PAM application rate (to 20 kg ha(-1)) also reduced sediment loss. The efficacy of this practice decreases slightly with successive rainfall events but still had significant benefit through four simulated rainfalls on soil packed into boxes. This was not the case in the field where the effect of PAM was limited to the first two rainfall events. In general, runoff volume was not strongly influenced by any of these practices, and most of the P loss was comprised of sediment-bound P. Both conservation practices are effective at reducing soil and nutrient loss in intensive potato systems.
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Increased susceptibility of Trpv4-deficient mice to obesity and obesity-induced osteoarthritis with very high-fat diet.
Ann. Rheum. Dis.
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To test the hypotheses that: (1) the transient receptor potential vanilloid 4 (TRPV4) ion channel is protective in the obesity model of osteoarthritis (OA), resulting in more severe obesity-induced OA in Trpv4 knockout (Trpv4(-/-)) mice; and (2) loss of TRPV4 alters mesodermal stem cell differentiation.
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Fibroblast growth factor receptor 1 activation in mammary tumor cells promotes macrophage recruitment in a CX3CL1-dependent manner.
PLoS ONE
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Tumor formation is an extensive process requiring complex interactions that involve both tumor cell-intrinsic pathways and soluble mediators within the microenvironment. Tumor cells exploit the intrinsic functions of many soluble molecules, including chemokines and their receptors, to regulate pro-tumorigenic phenotypes that are required for growth and progression of the primary tumor. Previous studies have shown that activation of inducible FGFR1 (iFGFR1) in mammary epithelial cells resulted in increased proliferation, migration, and invasion in vitro and tumor formation in vivo. These studies also demonstrated that iFGFR1 activation stimulated recruitment of macrophages to the epithelium where macrophages contributed to iFGFR1-mediated epithelial cell proliferation and angiogenesis. The studies presented here further utilize this model to identify the mechanisms that regulate FGFR1-induced macrophage recruitment. Results from this study elucidate a novel role for the inflammatory chemokine CX3CL1 in FGFR1-induced macrophage migration. Specifically, we illustrate that activation of both the inducible FGFR1 construct in mouse mammary epithelial cells and endogenous FGFR in the triple negative breast cancer cell line, HS578T, leads to expression of the chemokine CX3CL1. Furthermore, we demonstrate that FGFR-induced CX3CL1 is sufficient to recruit CX3CR1-expressing macrophages in vitro. Finally, blocking CX3CR1 in vivo leads to decreased iFGFR1-induced macrophage recruitment, which correlates with decreased angiogenesis. While CX3CL1 is a known target of FGF signaling in the wound healing environment, these studies demonstrate that FGFR activation also leads to induction of CX3CL1 in a tumor setting. Furthermore, these results define a novel role for CX3CL1 in promoting macrophage recruitment during mammary tumor formation, suggesting that the CX3CL1/CX3CR1 axis may represent a potential therapeutic approach for targeting breast cancers associated with high levels of tumor-associated macrophages.
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Online nanoscale ERLIC-MS outperforms RPLC-MS for shotgun proteomics in complex mixtures.
J. Proteome Res.
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We have explored the use of electrostatic repulsion hydrophilic interaction chromatography (ERLIC) as an alternative to the gold-standard in shotgun proteomics: reversed-phase (RP) LC for online ESI-MS/MS. Conditions for sample solubilization and initial gradient conditions were optimized to strike a balance between peptide solubility and maximum peptide retention when using mobile phase with high organic solvent concentration. Online ERLIC-MS demonstrated a 57% increase in total peptide identifications compared to RP-MS. We examined the mechanism of this improved performance and found that it stems from ERLICs propensity to retain longer peptides, which can be identified with greater confidence. Online nanoscale ERLIC-MS provides a powerful new tool for enhancing MS-based shotgun proteomic in a broad range of applications.
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Protein carbonylation and adipocyte mitochondrial function.
J. Biol. Chem.
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Carbonylation is the covalent, non-reversible modification of the side chains of cysteine, histidine, and lysine residues by lipid peroxidation end products such as 4-hydroxy- and 4-oxononenal. In adipose tissue the effects of such modifications are associated with increased oxidative stress and metabolic dysregulation centered on mitochondrial energy metabolism. To address the role of protein carbonylation in the pathogenesis of mitochondrial dysfunction, quantitative proteomics was employed to identify specific targets of carbonylation in GSTA4-silenced or overexpressing 3T3-L1 adipocytes. GSTA4-silenced adipocytes displayed elevated carbonylation of several key mitochondrial proteins including the phosphate carrier protein, NADH dehydrogenase 1? subcomplexes 2 and 3, translocase of inner mitochondrial membrane 50, and valyl-tRNA synthetase. Elevated protein carbonylation is accompanied by diminished complex I activity, impaired respiration, increased superoxide production, and a reduction in membrane potential without changes in mitochondrial number, area, or density. Silencing of the phosphate carrier or NADH dehydrogenase 1? subcomplexes 2 or 3 in 3T3-L1 cells results in decreased basal and maximal respiration. These results suggest that protein carbonylation plays a major instigating role in cytokine-dependent mitochondrial dysfunction and may be linked to the development of insulin resistance in the adipocyte.
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A novel mouse running wheel that senses individual limb forces: biomechanical validation and in vivo testing.
J. Appl. Physiol.
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Biomechanical data provide fundamental information about changes in musculoskeletal function during development, adaptation, and disease. To facilitate the study of mouse locomotor biomechanics, we modified a standard mouse running wheel to include a force-sensitive rung capable of measuring the normal and tangential forces applied by individual paws. Force data were collected throughout the night using an automated threshold trigger algorithm that synchronized force data with wheel-angle data and a high-speed infrared video file. During the first night of wheel running, mice reached consistent running speeds within the first 40 force events, indicating a rapid habituation to wheel running, given that mice generated >2,000 force-event files/night. Average running speeds and peak normal and tangential forces were consistent throughout the first four nights of running, indicating that one night of running is sufficient to characterize the locomotor biomechanics of healthy mice. Twelve weeks of wheel running significantly increased spontaneous wheel-running speeds (16 vs. 37 m/min), lowered duty factors (ratio of foot-ground contact time to stride time; 0.71 vs. 0.58), and raised hindlimb peak normal forces (93 vs. 115% body wt) compared with inexperienced mice. Peak normal hindlimb-force magnitudes were the primary force component, which were nearly tenfold greater than peak tangential forces. Peak normal hindlimb forces exceed the vertical forces generated during overground running (50-60% body wt), suggesting that wheel running shifts weight support toward the hindlimbs. This force-instrumented running-wheel system provides a comprehensive, noninvasive screening method for monitoring gait biomechanics in mice during spontaneous locomotion.
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Pathobiology of obesity and osteoarthritis: integrating biomechanics and inflammation.
Pathobiol Aging Age Relat Dis
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Obesity is a significant risk factor for developing osteoarthritis in weight-bearing and non-weight-bearing joints. Although the pathogenesis of obesity-associated osteoarthritis is not completely understood, recent studies indicate that pro-inflammatory metabolic factors contribute to an increase in osteoarthritis risk. Adipose tissue, and in particular infrapatellar fat, is a local source of pro-inflammatory mediators that are increased with obesity and have been shown to increase cartilage degradation in cell and tissue culture models. One adipokine in particular, leptin, may be a critical mediator of obesity-associated osteoarthritis via synergistic actions with other inflammatory cytokines. Biomechanical factors may also increase the risk of osteoarthritis by activating cellular inflammation and promoting oxidative stress. However, some types of biomechanical stimulation, such as physiologic cyclic loading, inhibit inflammation and protect against cartilage degradation. A high percentage of obese individuals with knee osteoarthritis are sedentary, suggesting that a lack of physical activity may increase the susceptibility to inflammation. A more comprehensive approach to understanding how obesity alters daily biomechanical exposures within joint tissues may provide new insight into the protective and damaging effects of biomechanical factors on inflammation in osteoarthritis.
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Workflow for analysis of high mass accuracy salivary data set using MaxQuant and ProteinPilot search algorithm.
Proteomics
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LTQ Orbitrap data analyzed with ProteinPilot can be further improved by MaxQuant raw data processing, which utilizes precursor-level high mass accuracy data for peak processing and MGF creation. In particular, ProteinPilot results from MaxQuant-processed peaklists for Orbitrap data sets resulted in improved spectral utilization due to an improved peaklist quality with higher precision and high precursor mass accuracy (HPMA). The output and postsearch analysis tools of both workflows were utilized for previously unexplored features of a three-dimensional fractionated and hexapeptide library (ProteoMiner) treated whole saliva data set comprising 200 fractions. ProteinPilots ability to simultaneously predict multiple modifications showed an advantage from ProteoMiner treatment for modified peptide identification. We demonstrate that complementary approaches in the analysis pipeline provide comprehensive results for the whole saliva data set acquired on an LTQ Orbitrap. Overall our results establish a workflow for improved protein identification from high mass accuracy data.
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A functional screen provides evidence for a conserved, regulatory, juxtamembrane phosphorylation site in guanylyl cyclase a and B.
PLoS ONE
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Kinase homology domain (KHD) phosphorylation is required for activation of guanylyl cyclase (GC)-A and -B. Phosphopeptide mapping identified multiple phosphorylation sites in GC-A and GC-B, but these approaches have difficulty identifying sites in poorly detected peptides. Here, a functional screen was conducted to identify novel sites. Conserved serines or threonines in the KHDs of phosphorylated receptor GCs were mutated to alanine and tested for reduced hormone to detergent activity ratios. Mutation of Ser-489 in GC-B to alanine but not glutamate reduced the activity ratio to 60% of wild type (WT) levels. Similar results were observed with Ser-473, the homologous site in GC-A. Receptors containing glutamates for previously identified phosphorylation sites (GC-A-6E and GC-B-6E) were activated to ~20% of WT levels but the additional glutamate substitution for S473 or S489 increased activity to near WT levels. Substrate-velocity assays indicated that GC-B-WT-S489E and GC-B-6E-S489E had lower Km values and that WT-GC-B-S489A, GC-B-6E and GC-B-6E-S489A had higher Km values than WT-GC-B. Homologous desensitization was enhanced when GC-A contained the S473E substitution, and GC-B-6E-S489E was resistant to inhibition by a calcium elevating treatment or protein kinase C activation--processes that dephosphorylate GC-B. Mass spectrometric detection of a synthetic phospho-Ser-473 containing peptide was 200-1300-fold less sensitive than other phosphorylated peptides and neither mass spectrometric nor (32)PO(4) co-migration studies detected phospho-Ser-473 or phospho-Ser-489 in cells. We conclude that Ser-473 and Ser-489 are Km-regulating phosphorylation sites that are difficult to detect using current methods.
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SH3BP4 is a negative regulator of amino acid-Rag GTPase-mTORC1 signaling.
Mol. Cell
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Amino acids stimulate cell growth and suppress autophagy through activation of mTORC1. The activation of mTORC1 by amino acids is mediated by Rag guanosine triphosphatase (GTPase) heterodimers on the lysosome. The molecular mechanism by which amino acids regulate the Rag GTPase heterodimers remains to be elucidated. Here, we identify SH3 domain-binding protein 4 (SH3BP4) as a binding protein and a negative regulator of Rag GTPase complex. SH3BP4 binds to the inactive Rag GTPase complex through its Src homology 3 (SH3) domain under conditions of amino acid starvation and inhibits the formation of active Rag GTPase complex. As a consequence, the binding abrogates the interaction of mTORC1 with Rag GTPase complex and the recruitment of mTORC1 to the lysosome, thus inhibiting amino acid-induced mTORC1 activation and cell growth and promoting autophagy. These results demonstrate that SH3BP4 is a negative regulator of the Rag GTPase complex and amino acid-dependent mTORC1 signaling.
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Deep metaproteomic analysis of human salivary supernatant.
Proteomics
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The human salivary proteome is extremely complex, including proteins from salivary glands, serum, and oral microbes. Much has been learned about the host component, but little is known about the microbial component. Here we report a metaproteomic analysis of salivary supernatant pooled from six healthy subjects. For deep interrogation of the salivary proteome, we combined protein dynamic range compression (DRC), multidimensional peptide fractionation, and high-mass accuracy MS/MS with a novel two-step peptide identification method using a database of human proteins plus those translated from oral microbe genomes. Peptides were identified from 124 microbial species as well as uncultured phylotypes such as TM7. Streptococcus, Rothia, Actinomyces, Prevotella, Neisseria, Veilonella, Lactobacillus, Selenomonas, Pseudomonas, Staphylococcus, and Campylobacter were abundant among the 65 genera from 12 phyla represented. Taxonomic diversity in our study was broadly consistent with metagenomic studies of saliva. Proteins mapped to 20 KEGG pathways, with carbohydrate metabolism, amino acid metabolism, energy metabolism, translation, membrane transport, and signal transduction most represented. The communities sampled appear to be actively engaged in glycolysis and protein synthesis. This first deep metaproteomic catalog from human salivary supernatant provides a baseline for future studies of shifts in microbial diversity and protein activities potentially associated with oral disease.
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Links among nitrification, nitrifier communities, and edaphic properties in contrasting soils receiving dairy slurry.
J. Environ. Qual.
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Soil biotic and abiotic factors strongly influence nitrogen (N) availability and increases in nitrification rates associated with the application of manure. In this study, we examine the effects of edaphic properties and a dairy (Bos taurus) slurry amendment on N availability, nitrification rates and nitrifier communities. Soils of variable texture and clay mineralogy were collected from six USDA-ARS research sites and incubated for 28 d with and without dairy slurry applied at a rate of ~300 kg N ha(-1). Periodically, subsamples were removed for analyses of 2 M KCl extractable N and nitrification potential, as well as gene copy numbers of ammonia-oxidizing bacteria (AOB) and archaea (AOA). Spearman coefficients for nitrification potentials and AOB copy number were positively correlated with total soil C, total soil N, cation exchange capacity, and clay mineralogy in treatments with and without slurry application. Our data show that the quantity and type of clay minerals present in a soil affect nitrifier populations, nitrification rates, and the release of inorganic N. Nitrogen mineralization, nitrification potentials, and edaphic properties were positively correlated with AOB gene copy numbers. On average, AOA gene copy numbers were an order of magnitude lower than those of AOB across the six soils and did not increase with slurry application. Our research suggests that the two nitrifier communities overlap but have different optimum environmental conditions for growth and activity that are partly determined by the interaction of manure-derived ammonium with soil properties.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.