Sample Stability and Protein Composition of Saliva: Implications for Its Use as a Diagnostic Fluid

Saliva is an easy accessible plasma ultra-filtrate. Therefore, saliva can be an attractive alternative to blood for measurement of diagnostic protein markers. Our aim was to determine stability and protein composition of saliva. Protein stability at room temperature was examined by incubating fresh whole saliva with and without inhibitors of proteases and bacterial metabolism followed by Surface Enhanced Laser Desorption/Ionization (SELDI) analyses. Protein composition was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) fractionation of saliva proteins followed by digestion of excised bands and identification by liquid chromatography tandem mass spectrometry (LC-MS/MS). Results show that rapid protein degradation occurs within 30 minutes after sample collection. Degradation starts already during collection. Protease inhibitors partly prevented degradation while inhibition of bacterial metabolism did not affect degradation. Three stable degradation products of 2937 Da, 3370 Da and 4132 Da were discovered which can be used as markers to monitor sample quality. Saliva proteome analyses revealed 218 proteins of which 84 can also be found in blood plasma. Based on a comparison with seven other proteomics studies on whole saliva we identified 83 new saliva proteins. We conclude that saliva is a promising diagnostic fluid when precautions are taken towards protein breakdown

Comparative analysis of the human hepatic and adipose tissue transcriptomes during LPS-induced inflammation leads to the identification of differential biological pathways and candidate biomarkers

<p>Abstract</p> <p>Background</p> <p>Insulin resistance (IR) is accompanied by chronic low grade systemic inflammation, obesity, and deregulation of total body energy homeostasis. We induced inflammation in adipose and liver tissues <it>in vitro </it>in order to mimic inflammation <it>in vivo </it>with the aim to identify tissue-specific processes implicated in IR and to find biomarkers indicative for tissue-specific IR.</p> <p>Methods</p> <p>Human adipose and liver tissues were cultured in the absence or presence of LPS and DNA Microarray Technology was applied for their transcriptome analysis. Gene Ontology (GO), gene functional analysis, and prediction of genes encoding for secretome were performed using publicly available bioinformatics tools (DAVID, STRING, SecretomeP). The transcriptome data were validated by proteomics analysis of the inflamed adipose tissue secretome.</p> <p>Results</p> <p>LPS treatment significantly affected 667 and 483 genes in adipose and liver tissues respectively. The GO analysis revealed that during inflammation adipose tissue, compared to liver tissue, had more significantly upregulated genes, GO terms, and functional clusters related to inflammation and angiogenesis. The secretome prediction led to identification of 399 and 236 genes in adipose and liver tissue respectively. The secretomes of both tissues shared 66 genes and the remaining genes were the differential candidate biomarkers indicative for inflamed adipose or liver tissue. The transcriptome data of the inflamed adipose tissue secretome showed excellent correlation with the proteomics data.</p> <p>Conclusions</p> <p>The higher number of altered proinflammatory genes, GO processes, and genes encoding for secretome during inflammation in adipose tissue compared to liver tissue, suggests that adipose tissue is the major organ contributing to the development of systemic inflammation observed in IR. The identified tissue-specific functional clusters and biomarkers might be used in a strategy for the development of tissue-targeted treatment of insulin resistance in patients.</p

Comparison of Isotope-labeled Amino Acid Incorporation Rates (CILAIR) Provides a Quantitative Method to Study Tissue Secretomes

Adipose tissue is an endocrine organ involved in regulation of whole-body energy metabolism via storage of lipids and secretion of various peptide hormones (adipokines). We previously characterized the adipose tissue secretome and showed that [(13)C]lysine incorporation into secreted proteins can be used to determine the origin of identified proteins. In the present study we determined the effect of insulin on the secretome by comparing incorporation rates of (13)C-labeled lysine in the presence and absence of insulin. Human visceral adipose tissue from one patient was divided over six dishes. After subsequent washes to remove serum proteins, [(13)C]lysinecontaining medium was added. Three dishes also received 60 nm insulin. The other three were controls. After 72 h of culture, media were collected and processed separately, involving concentration by ultrafiltration and fractionation by SDS-PAGE followed by in-gel digestion of excised bands and LC-MS/MS analyses. The obtained spectra were used for database searching and calculation of heavy/light ratios. The three control data sets shared 342 proteins of which 156 were potentially secreted and contained label. The three insulin-derived data sets shared 361 proteins of which 141 were potentially secreted and contained label. After discarding secreted proteins with very low label incorporation, 121 and 113 proteins remained for control and insulin data sets, respectively. The average coefficient of variation for control triplicates was 10.0% and for insulin triplicates was 18.3%. By comparing heavy/light ratios in the absence and presence of insulin we found 24 up-regulated proteins and four down-regulated proteins, and 58 proteins showed no change. Proteins involved in the endoplasmic reticulum stress response and in extracellular matrix remodeling were up-regulated by insulin. In conclusion, comparison of isotope-labeled amino acid incorporation rates (CILAIR) allows quantitative assessment of changes in protein secretion without the need for 100% label incorporation, which cannot be reached in differentiated tissues or cells. Molecular & Cellular Proteomics 8:316-324, 2009

Quantitative proteomics analyses of activation states of human THP-1 macrophages

Macrophages display large functional and phenotypical plasticity. They can adopt a broad range of activation states depending on their microenvironment Various surface markers are used to characterize these differentially polarized macrophages. However, this is not informative for the functions of the macrophage. In order to have a better understanding of the functional changes of macrophages upon differential polarization, we studied differences in LPS- and IL4-stimulated macrophages. The THP-1 human monocytic cell line, was used as a model system. Cells were labeled, differentiated and stimulated with either LPS or IL-4 in a quantitative SILAC proteomics set-up. The resulting sets of proteins were functionally clustered. LPS-stimulated macrophages show increased secretion of proinflammatoly peptides, leading to increased pressure on protein biosynthesis and processing. IL4-stimulated macrophages show upregulation of cell adhesion and extracellular matrix remodeling. Our approach provides an integrated view of polarization-induced functional changes and proves useful for studying functional differences between subsets of macrophages. Moreover, the identified polarization specific proteins may contribute to a better characterization of different activation states in situ and their role in various inflammatory processes. (C) 2015 Elsevier B.V. All rights reserved

Characterization of the human visceral adipose tissue secretome

Adipose tissue is an endocrine organ involved in storage and release of energy but also in regulation of energy metabolism in other organs via secretion of peptide and protein hormones (adipokines). Especially visceral adipose tissue has been implicated in the development of metabolic syndrome and type 2 diabetes. Factors secreted by the stromal-vascular fraction contribute to the secretome and modulate adipokine secretion by adipocytes. Therefore, we aimed at the characterization of the adipose tissue secretome rather than the adipocyte cell secretome. The presence of serum proteins and intracellular proteins from damaged cells, released during culture, may dramatically influence the dynamic range of the sample and thereby identification of secreted proteins. Part of the study was therefore dedicated to the influence of the culture setup on the quality of the final sample. Visceral adipose tissue was cultured in five experimental setups, and the quality of resulting samples was evaluated in terms of protein concentration and protein composition. The best setup involved one wash after the 1st h in culture followed by two or three additional washes within an 8-h period, starting after overnight culture. Thereafter tissue was maintained in culture for an additional 48-114 h to obtain the final sample. For the secretome experiment, explants were cultured in media containing L-[C-13(6),N-15(2)]lysine to validate the origin of the identified proteins (adipose tissue- or serum-derived). In total, 259 proteins were identified with >= 99% confidence. 108 proteins contained a secretion signal peptide of which 70 incorporated the label and were considered secreted by adipose tissue. These proteins were classified into five categories according to function. This is the first study on the (human) adipose tissue secretome. The results of this study contribute to a better understanding of the role of adipose tissue in whole body energy metabolism and related diseases

Factors related to colonic fermentation of nondigestible carbohydrates of a previous evening meal increase tissue glucose uptake and moderate glucose-associated inflammation

&lt;b&gt;Background&lt;/b&gt;: Evening meals that are rich in nondigestible carbohydrates have been shown to lower postprandial glucose concentrations after ingestion of high-glycemic-index breakfasts. This phenomenon is linked to colonic fermentation of nondigestible carbohydrates, but the underlying mechanism is not fully elucidated. &lt;b&gt;Objective&lt;/b&gt;: We examined the way in which glucose kinetics and related factors change after breakfast as a result of colonic fermentation. &lt;b&gt;Design&lt;/b&gt;: In a crossover design, 10 healthy men ingested as an evening meal white wheat bread (WB) or cooked barley kernels (BA) that were rich in nondigestible carbohydrates. In the morning after intake of 50 g &lt;sup&gt;13&lt;/sup&gt;C-enriched glucose, the dual-isotope technique was applied to determine glucose kinetics. Plasma insulin, free fatty acid, interleukin-6, tumor necrosis factor-{alpha}, and short-chain fatty acid concentrations and breath-hydrogen excretion were measured. &lt;b&gt;Results&lt;/b&gt;: The plasma glucose response after the glucose drink was 29% lower after the BA evening meal (P = 0.019). The insulin response was the same, whereas mean (±SEM) tissue glucose uptake was 30% higher (20.2 ± 1.9 compared with 15.5 ± 1.8 mL/2 h; P = 0.016) after the BA evening meal, which indicated higher peripheral insulin sensitivity (P = 0.001). The 4-h mean postprandial interleukin-6 (19.7 ± 5.1 compared with 5.1 ± 0.7 pg/mL; P = 0.024) and tumor necrosis factor-&#945; (7.8 ± 2.1 compared with 5.3 ± 1.6 pg/mL; P = 0.008) concentrations after the glucose drink were higher after the WB evening meal. Butyrate concentrations (P = 0.041) and hydrogen excretion (P = 0.005) were higher in the morning after the BA evening meal. &lt;b&gt;Conclusion&lt;/b&gt;: In healthy subjects, factors related to colonic fermentation of nondigestible carbohydrates increase peripheral insulin sensitivity and moderate glucose-associated inflammation

Integrative Kinome Profiling Identifies mTORC1/2 Inhibition as Treatment Strategy in Ovarian Clear Cell Carcinoma

Purpose: Advanced-stage ovarian clear cell carcinoma (OCCC) is unresponsive to conventional platinum-based chemotherapy. Frequent alterations in OCCC include deleterious mutations in the tumor suppressor ARID1A and activating mutations in the PI3K subunit PIK3CA In this study, we aimed to identify currently unknown mutated kinases in patients with OCCC and test druggability of downstream affected pathways in OCCC models.Experimental Design: In a large set of patients with OCCC (n = 124), the human kinome (518 kinases) and additional cancer-related genes were sequenced, and copy-number alterations were determined. Genetically characterized OCCC cell lines (n = 17) and OCCC patient-derived xenografts (n = 3) were used for drug testing of ERBB tyrosine kinase inhibitors erlotinib and lapatinib, the PARP inhibitor olaparib, and the mTORC1/2 inhibitor AZD8055.Results: We identified several putative driver mutations in kinases at low frequency that were not previously annotated in OCCC. Combining mutations and copy-number alterations, 91% of all tumors are affected in the PI3K/AKT/mTOR pathway, the MAPK pathway, or the ERBB family of receptor tyrosine kinases, and 82% in the DNA repair pathway. Strong p-S6 staining in patients with OCCC suggests high mTORC1/2 activity. We consistently found that the majority of OCCC cell lines are especially sensitive to mTORC1/2 inhibition by AZD8055 and not toward drugs targeting ERBB family of receptor tyrosine kinases or DNA repair signaling. We subsequently demonstrated the efficacy of mTORC1/2 inhibition in all our unique OCCC patient-derived xenograft models.Conclusions: These results propose mTORC1/2 inhibition as an effective treatment strategy in OCCC. Clin Cancer Res; 24(16); 3928-40. ©2018 AACR.status: publishe