Side-Chain Immune Oxysterols Induce Neuroinflammation by Activating Microglia.

In individuals with Alzheimer's disease, the brain exhibits elevated levels of IL-1ß and oxygenated cholesterol molecules (oxysterols). This study aimed to investigate the effects of side-chain oxysterols on IL-1ß expression using HMC3 microglial cells and ApoE-deficient mice. Treatment of HMC3 cells with 25-hydroxycholesterol (25OHChol) and 27-hydroxycholesterol (27OHChol) led to increased IL-1ß expression at the transcript and protein levels. Additionally, these oxysterols upregulated the surface expression of MHC II, a marker of activated microglia. Immunohistochemistry performed on the mice showed increased microglial expression of IL-1ß and MHC II when fed a high-cholesterol diet. However, cholesterol and 24s-hydroxycholesterol did not increase IL-1ß transcript levels or MHC II expression. The extent of IL-1ß increase induced by 25OHChol and 27OHChol was comparable to that caused by oligomeric ß-amyloid, and the IL-1ß expression induced by the oxysterols was not impaired by polymyxin B, which inhibited lipopolysaccharide-induced IL-1ß expression. Both oxysterols enhanced the phosphorylation of Akt, ERK, and Src, and inhibition of these kinase pathways with pharmacological inhibitors suppressed the expression of IL-1ß and MHC II. The pharmacological agents chlorpromazine and cyclosporin A also impaired the oxysterol-induced expression of IL-1ß and upregulation of MHC II. Overall, these findings suggest that dysregulated cholesterol metabolism leading to elevated levels of side-chain oxysterols, such as 25OHChol and 27OHChol, can activate microglia to secrete IL-1ß through a mechanism amenable to pharmacologic intervention. The activation of microglia and subsequent neuroinflammation elicited by the immune oxysterols can contribute to the development of neurodegenerative diseases.

Analysis of significant protein abundance from multiple reaction-monitoring data.

BACKGROUND: Discovering reliable protein biomarkers is one of the most important issues in biomedical research. The ELISA is a traditional technique for accurate quantitation of well-known proteins. Recently, the multiple reaction-monitoring (MRM) mass spectrometry has been proposed for quantifying newly discovered protein and has become a popular alternative to ELISA. For the MRM data analysis, linear mixed modeling (LMM) has been used to analyze MRM data. MSstats is one of the most widely used tools for MRM data analysis that is based on the LMMs. However, LMMs often provide various significance results, depending on model specification. Sometimes it would be difficult to specify a correct LMM method for the analysis of MRM data. Here, we propose a new logistic regression-based method for Significance Analysis of Multiple Reaction Monitoring (LR-SAM). RESULTS: Through simulation studies, we demonstrate that LMM methods may not preserve type I error, thus yielding high false- positive errors, depending on how random effects are specified. Our simulation study also shows that the LR-SAM approach performs similarly well as LMM approaches, in most cases. However, LR-SAM performs better than the LMMs, particularly when the effects sizes of peptides from the same protein are heterogeneous. Our proposed method was applied to MRM data for identification of proteins associated with clinical responses of treatment of 115 hepatocellular carcinoma (HCC) patients with the tyrosine kinase inhibitor sorafenib. Of 124 candidate proteins, LMM approaches provided 6 results varying in significance, while LR-SAM, by contrast, yielded 18 significant results that were quite reproducibly consistent. CONCLUSION: As exemplified by an application to HCC data set, LR-SAM more effectively identified proteins associated with clinical responses of treatment than LMM did.

Serum fibronectin distinguishes the early stages of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death, necessitating the discovery of serum markers for its early detection. In this study, a total of 180 serum samples from liver cirrhosis (LC), hepatocellular carcinoma (HCC) patients and paired samples of HCC patients who recovered (Recovery) were analyzed by multiple reaction monitoring-mass spectrometry (MRM-MS) to verify biomarkers. The three-fold crossvalidation was repeated 100 times in the training and test sets to evaluate statistical significance of 124 candidate proteins. This step resulted in 2 proteins that had an area under the receiver operating curve (AUROC) values ≥0.800 in the training (n = 90) and test sets (n = 90). Specifically, fibronectin (FN1, WCGTTQNYDADQK), distinguished HCC from LC patients, with an AUROC value of 0.926 by logistic regression. A FN1 protein was selected for validation in an independent sample (n = 60) using enzyme-linked immunosorbent assay (ELISA). The combination of alpha-fetoprotein (AFP) and FN1 improved the diagnostic performance and differentiated HCC patients with normal AFP levels. Our study has examined candidate markers for the benign disease state and malignancy and has followed up on the consequent recovery. Thus, improvement in the early detection of HCC by a 2-marker panel (AFP + FN1) might benefit HCC patients.

Prediction of Response to Sorafenib in Hepatocellular Carcinoma: A Putative Marker Panel by Multiple Reaction Monitoring-Mass Spectrometry (MRM-MS).

Sorafenib is the only standard treatment for unresectable hepatocellular carcinoma (HCC), but it provides modest survival benefits over placebo, necessitating predictive biomarkers of the response to sorafenib. Serum samples were obtained from 115 consecutive patients with HCC before sorafenib treatment and analyzed by multiple reaction monitoring-mass spectrometry (MRM-MS) and ELISA to quantify candidate biomarkers. We verified a triple-marker panel to be predictive of the response to sorafenib by MRM-MS, comprising CD5 antigen-like (CD5L), immunoglobulin J (IGJ), and galectin-3-binding protein (LGALS3BP), in HCC patients. This panel was a significant predictor (AUROC > 0.950) of the response to sorafenib treatment, having the best cut-off value (0.4) by multivariate analysis. In the training set, patients who exceeded this cut-off value had significantly better overall survival (median, 21.4 months) than those with lower values (median, 8.6 months; p = 0.001). Further, a value that was lower than this cutoff was an independent predictor of poor overall survival [hazard ratio (HR), 2.728; 95% confidence interval (CI), 1.312-5.672; p = 0.007] and remained an independent predictive factor of rapid progression (HR, 2.631; 95% CI, 1.448-4.780; p = 0.002). When applied to the independent validation set, levels of the cut-off value for triple-marker panel maintained their prognostic value for poor clinical outcomes. On the contrast, the triple-marker panel was not a prognostic factor for patients who were treated with transarterial chemoembolization (TACE). The discriminatory signature of a triple-marker panel provides new insights into targeted proteomic biomarkers for individualized sorafenib therapy.

Isolation and Characterization of Pepsin-soluble Collagens from Bones, Skins, and Tendons in Duck Feet.

The objectives of this study were conducted to characterize pepsin-soluble collagen (PSC) extracted from bones (PSC-B), skins (PSC-S), and tendons (PSC-T) of duck feet and to determine their thermal and structural properties, for better practical application of each part of duck feet as a novel source for collagen. PSC was extracted from each part of duck feet by using 0.5 M acetic acid containing 5% (w/w) pepsin. Electrophoretic patterns showed that the ratio between α1 and α2 chains, which are subunit polypeptides forming collagen triple helix, was approximately 1:1 in all PSCs of duck feet. PSC-B had slightly higher molecular weights for α1 and α2 chains than PSC-S and PSC-T. From the results of differential scanning calorimetry (DSC), higher onset (beginning point of melting) and peak temperatures (maximum point of curve) were found at PSC-B compared to PSC-S and PSC-T (p<0.05). Fourier transform infrared spectroscopy (FT-IR) presented that PSC-S and PSC-T had similar intermolecular structures and chemical bonds, whereas PSC-B exhibited slight difference in amide A region. Irregular dense sheet-like films linked by random-coiled filaments were observed similarly. Our findings indicate that PSCs of duck feet might be characterized similarly as a mixture of collagen type I and II and suggest that duck feet could be used for collagen extraction without deboning and/or separation processes.

Effect of Ginger Extract and Citric Acid on the Tenderness of Duck Breast Muscles.

The objective of this study was to examine the effect of ginger extract (GE) combined with citric acid on the tenderness of duck breast muscles. Total six marinades were prepared with the combination of citric acid (0 and 0.3 M citric acid) and GE (0, 15, and 30%). Each marinade was sprayed on the surface of duck breasts (15 mL/100 g), and the samples were marinated for 72 h at 4℃. The pH and proteolytic activity of marinades were determined. After 72 h of marination, Warner Bratzler shear force (WBSF), myofibrillar fragmentation index (MFI), pH, cooking loss, moisture content, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein solubility were evaluated. There was no significant (p>0.05) difference in moisture content or cooking loss among all samples. However, GE marination resulted in a significant (p<0.05) decrease in WBSF but a significant (p<0.05) increase in pH and MFI. In addition, total protein and myofibrillar protein solubility of GE-marinated duck breast muscles in both WOC (without citric acid) and WC (with citric acid) conditions were significantly (p<0.05) increased compared to non-GE-marinated duck breast muscles. SDS-PAGE showed an increase of protein degradation (MHC and actin) in WC condition compared to WOC condition. There was a marked actin reduction in GE-treated samples in WC. The tenderization effect of GE combined with citric acid may be attributed to various mechanisms such as increased MFI and myofibrillar protein solubility.

Effects of Red and Green Glassworts (Salicornia herbacea L.) on Physicochemical and Textural Properties of Reduced-salt Cooked Sausages.

This study was conducted to determine the effects of red and green glasswort on the physicochemical and textural properties of reduced-salt cooked sausages. The control was formulated with 1.5% NaCl; then, three reduced-salt treatments were prepared, with 0.75% NaCl (RS), 0.75% NaCl+1.0% red glasswort (RSR) and 0.75% NaCl+1.0% green glasswort (RSG), respectively. The addition of glasswort within the added amount of 1% had no influence on the pH value of the reduced-salt cooked sausages, regardless of the glasswort type. In terms of color, RSG treatment conveyed a higher hue angle value than the RSR treatment (p<0.05). Increases in the protein solubility (total and myofibrillar proteins) and apparent viscosity of reduced-salt meat batter that were due to the addition of glasswort were observed; however, there were no differences according to the type of glasswort (p>0.05). Furthermore, the addition of glasswort, regardless of its type, resulted in decreased cooking loss, and increased emulsion stability. As a result, reduced-salt cooked sausages formulated with either red or green glasswort demonstrated similar textural properties to those of the control. In conclusion, the type of glasswort within an added amount of 1% had no influence on the physicochemical and textural properties of reduced-salt cooked sausages, except for the color characteristics. In terms of color alteration by the addition of glasswort, the red glasswort, which in comparison with the green glasswort could minimize the color changes of reduced-salt cooked sausages, might be an effective source for manufacturing meat products.