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1.
Am J Physiol Heart Circ Physiol ; 311(1): H219-28, 2016 07 01.
Article in English | MEDLINE | ID: mdl-27199111

ABSTRACT

Autophagy is regulated by nutrient and energy status and plays an adaptive role during nutrient deprivation and ischemic stress. Metabolic syndrome (MetS) is a hypernutritive state characterized by obesity, dyslipidemia, elevated fasting blood glucose levels, and insulin resistance. It has also been associated with impaired autophagic flux and larger-sized infarcts. We hypothesized that diet-induced obesity (DIO) affects nutrient sensing, explaining the observed cardiac impaired autophagy. We subjected male friend virus B NIH (FVBN) mice to a high-fat diet, which resulted in increased weight gain, fat deposition, hyperglycemia, insulin resistance, and larger infarcts after myocardial ischemia-reperfusion. Autophagic flux was impaired after 4 wk on a high-fat diet. To interrogate nutrient-sensing pathways, DIO mice were subjected to overnight fasting, and hearts were processed for biochemical and proteomic analysis. Obese mice failed to upregulate LC3-II or to clear p62/SQSTM1 after fasting, although mRNA for LC3B and p62/SQSTM1 were appropriately upregulated in both groups, demonstrating an intact transcriptional response to fasting. Energy- and nutrient-sensing signal transduction pathways [AMPK and mammalian target of rapamycin (mTOR)] also responded appropriately to fasting, although mTOR was more profoundly suppressed in obese mice. Proteomic quantitative analysis of the hearts under fed and fasted conditions revealed broad changes in protein networks involved in oxidative phosphorylation, autophagy, oxidative stress, protein homeostasis, and contractile machinery. In many instances, the fasting response was quite discordant between lean and DIO mice. Network analysis implicated the peroxisome proliferator-activated receptor and mTOR regulatory nodes. Hearts of obese mice exhibited impaired autophagy, altered proteome, and discordant response to nutrient deprivation.


Subject(s)
Autophagy , Fasting/metabolism , Myocardial Infarction/metabolism , Myocardial Reperfusion Injury/metabolism , Myocardium/metabolism , Obesity/metabolism , AMP-Activated Protein Kinases/metabolism , Animals , Diet, High-Fat , Disease Models, Animal , Energy Metabolism , Male , Metabolic Syndrome/etiology , Metabolic Syndrome/metabolism , Mice , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Myocardial Infarction/etiology , Myocardial Infarction/physiopathology , Myocardial Reperfusion Injury/etiology , Myocardial Reperfusion Injury/pathology , Myocardium/pathology , Obesity/complications , Obesity/pathology , Peroxisome Proliferator-Activated Receptors/metabolism , Protein Interaction Maps , Proteolysis , Proteomics/methods , Sequestosome-1 Protein/genetics , Sequestosome-1 Protein/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Time Factors
2.
Proteomics ; 13(8): 1352-7, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23412978

ABSTRACT

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.


Subject(s)
Amino Acid Sequence , Databases, Protein , Peptides/chemistry , Proteomics/methods , Search Engine , Algorithms , Expressed Sequence Tags , Genomics/methods , Humans , Metagenome , Mouth Mucosa/metabolism , Saliva/metabolism , Sensitivity and Specificity , Software , Tandem Mass Spectrometry/methods
3.
Clin Proteomics ; 8: 13, 2011 Sep 13.
Article in English | MEDLINE | ID: mdl-21914210

ABSTRACT

INTRODUCTION: 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. METHODS: In this pilot study we evaluated the potential of a novel method using commercial PerioPaper absorbent strips for non-invasive collection of oral lesion exudate material coupled with mass spectrometry-based proteomics for oral cancer biomarker discovery. RESULTS: Our evaluation focused on three core issues. First, using an "on-strip" processing method, we found that protein can be isolated from exudate samples in amounts compatible with large-scale mass spectrometry-based proteomic analysis. Second, we found that the OPML exudate proteome was distinct from that of whole saliva, while being similar to the OPML epithelial cell proteome, demonstrating the fidelity of our exudate collection method. Third, in a proof-of-principle study, we identified numerous, inflammation-associated proteins showing an expected increase in abundance in OPML exudates compared to healthy oral tissue exudates. These results demonstrate the feasibility of identifying differentially abundant proteins from exudate samples, which is essential for biomarker discovery studies. CONCLUSIONS: Collectively, our findings demonstrate that our exudate collection method coupled with mass spectrometry-based proteomics has great potential for transforming OSCC biomarker discovery and clinical diagnostics assay development.

4.
PLoS One ; 5(6): e11148, 2010 Jun 17.
Article in English | MEDLINE | ID: mdl-20567502

ABSTRACT

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: To discover such proteins, in a first-of-its-kind study we used advanced mass spectrometry-based quantitative proteomics analysis of the pooled soluble fraction of whole saliva from four subjects with pre-malignant lesions and four with malignant lesions. We prioritized candidate biomarkers via bioinformatics and validated selected proteins by western blotting. Bioinformatic analysis of differentially abundant proteins and initial western blotting revealed increased abundance of myosin and actin in patients with malignant lesions. We validated those results by additional western blotting of individual whole saliva samples from twelve other subjects with pre-malignant oral lesions and twelve with malignant oral lesions. Sensitivity/specificity values for distinguishing between different lesion types were 100%/75% (p = 0.002) for actin, and 67%/83% (p<0.00001) for myosin in soluble saliva. Exfoliated epithelial cells from subjects' saliva also showed increased myosin and actin abundance in those with malignant lesions, linking our observations in soluble saliva to abundance differences between pre-malignant and malignant cells. CONCLUSIONS/SIGNIFICANCE: Salivary actin and myosin abundances distinguish oral lesion types with sensitivity and specificity rivaling other non-invasive oral cancer tests. Our findings provide a promising starting point for the development of non-invasive and inexpensive salivary tests to reliably detect oral cancer early.


Subject(s)
Actins/metabolism , Biomarkers/metabolism , Mouth Neoplasms/metabolism , Myosins/metabolism , Precancerous Conditions/metabolism , Proteomics , Chromatography, High Pressure Liquid , Humans , Mouth Neoplasms/diagnosis , Precancerous Conditions/diagnosis , Sensitivity and Specificity , Tandem Mass Spectrometry
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