Taxonomy: 2017

A disintegrin and metalloproteinase 17” (ADAM17), or tumour necrosis factor (TNF)-α converting enzyme, is an upstream target for mitigating TNF-α-mediated inflammation. ADAM17 can be inhibited by chelation of its catalytic site zinc cofactor, which is required for substrate catalysis and structure stabilization. In this study, rye secalin-derived tripeptides (CQV and QCA) and analogues (QCV and QVC) showed zinc-chelating capacity (∼35{8617e24ab0b76aabcd10cf8004a7bdc562123dc1ea8adc37299158a7c05423e6} at 0.5 μM) and dose-dependently inhibited ADAM17 activity with up to 70{8617e24ab0b76aabcd10cf8004a7bdc562123dc1ea8adc37299158a7c05423e6} inhibition were observed at 5 μM. Moreover, ADAM17 intrinsic fluorescence emission was quenched by the peptides via the dynamic mechanism, with CQV producing the highest quenching constants. Molecular docking revealed that the tripeptides interacted with ADAM17 active site residues, mostly occupying the S1 and S1′ subsites. CQV had the shortest distance to the zinc cofactor and lowest binding energy. The peptides coordinated zinc through their C-terminal carboxylate anions for QCV, QVC and CQV, and peptide bond carbonyl for CQV. CQV also had more hydrogen bonding with the N, O and H atoms of ADAM17 active site residues but, unlike the other peptides, this did not involve the peptidyl sulfhydryl groups. Interaction with ADAM17 S1′ hydrophobic pockets suggests a possible selectivity of the peptides for ADAM17. Despite their promise as bioactive candidates for controlling inflammation, incubation of THP-1 human monocytic cells with the tripeptide at a concentration that inhibited ADAM17 activity did not result in inhibition of lipopolysaccharide-stimulated TNF-α release.

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The global incidence of obesity has led to an increasing need for understanding the molecular mechanisms that drive this epidemic and its comorbidities. Quantitative real-time RT-PCR (RT-qPCR) is the most reliable and widely used method for gene expression analysis. The selection of suitable reference genes (RGs) is critical for obtaining accurate gene expression information. The current study aimed to identify optimal RGs to perform quantitative transcriptomic analysis based on RT-qPCR for obesity and diabetes research, employing in vitroand mouse models, and human tissue samples. Using the ReFinder program we evaluated the stability of a total of 15 RGs. The impact of choosing the most suitable RGs versus less suitable RGs on RT-qPCR results was assessed. Optimal RGs differed between tissue and cell type, species, and experimental conditions. By employing different sets of RGs to normalize the mRNA expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), we show that sub-optimal RGs can markedly alter the PGC1α gene expression profile. Our study demonstrates the importance of validating RGs prior to normalizing transcriptional expression levels of target genes and identifies optimal RG pairs for reliable RT-qPCR normalization in cells and in human and murine muscle and adipose tissue for obesity/diabetes research.

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BACKGROUND:

The characteristics of circulating inflammatory cells (leukocytes) in patients undergoing heart surgery remains poorly understood. Recently, neutrophil-to-lymphocyte ratio (NLR) and specific monocyte subsets (based on CD14/CD16 expression) have been suggested as markers of inflammation and predictors of outcomes. The present study aims to characterize the influence cardiac surgery with cardiopulmonary bypass has on specific circulating leukocytes.

METHODS:

All enrolled patients had blood samples taken pre- (0 days), early post- (5 days), and late post- (90 days) surgery. Complete blood counts were performed and whole leukocyte isolations were obtained from blood samples and analyzed with flow cytometry. Fluorophore-linked antibodies (CD45, CD11b, CD14, and CD16) were added to the blood cell isolations and later assessed by flow cytometry.

RESULTS:

Seventeen patients were enrolled and samples obtained at 0, 5, and 90 days. We demonstrated a significant increase in NLR (2.2-fold; p = 0.0028) and CD16 mean fluorescence index (MFI-measure fluorescence intensity shift of CD16 in a gated cell population) early at day 5 (2.0-fold; p = 0.0051). Both NLR and CD16 MFI levels generally returned to normal by day 90. There was a significant positive correlation between NLR and CD16 MFI (r² = 0.29; p = 0.0064). Adverse cardiovascular event (AE) was defined as prolonged length of hospitalization or readmission to hospital for cardiac reasons after discharge was seen in 59% of patients (no deaths occurred). In an unadjusted analysis of AE, we identified NLR as a likely predictor of AE, which meant that patients developing AE had a significantly higher baseline NLR (p = 0.0065), something that was not observed with CD16 MFI (p = 0.2541).

CONCLUSION:

Cardiac surgery is associated with a significant increase in NLR and CD16 MFI (non-classical monocytes) early after surgery corresponding to the early inflammatory phase after surgery. Furthermore, we have, for the first time, identified a significant correlation between NLR and CD16 MFI. While the mechanism for this relationship remains unclear, our findings support the use of a simple test of NLR as a biomarker of inflammation for predicting outcomes in cardiac surgery patients.

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