Differential effects of selenium and knock-down of glutathione peroxidases on TNFα and flagellin inflammatory responses in gut epithelial cells

Selenium (Se) is essential for human health. Despite evidence that Se intake affects inflammatory responses, the mechanisms by which Se and the selenoproteins modulate inflammatory signalling, especially in the gut, are not yet defined. The aim of this work was to assess effects of altered Se supply and knock-down of individual selenoproteins on NF-κB activation in gut epithelial cells. Caco-2 cells were stably transfected with gene constructs expressing luciferase linked either to three upstream NF-κB response elements and a TATA box or only a TATA box. TNFα and flagellin activated NF-κB-dependent luciferase activity and increased IL-8 expression. Se depletion decreased expression of glutathione peroxidase1 (GPX1) and selenoproteins H and W and increased TNFα-stimulated luciferase activity, endogenous IL-8 expression and reactive oxygen species (ROS) production. These effects were not mimicked by independent knock-down of either GPX1, selenoprotein H or W; indeed, GPX1 knock-down lowered TNFα-induced NF-κB activation and did not affect ROS levels. GPX4 knock-down decreased NF-κB activation by flagellin but not by TNFα. We hypothesise that Se depletion alters the pattern of expression of multiple selenoproteins that in turn increases ROS and modulates NF-κB activation in epithelial cells, but that the effect of GPX1 knock-down is ROS-independent

2D-electrophoresis and multiplex immunoassay proteomic analysis of different body fluids and cellular components reveal known and novel markers for extended fasting

Proteomic technologies applied for profiling human biofluids and blood cells are considered to reveal new biomarkers of exposure or provide insights into novel mechanisms of adaptation

Estimating trace deposition time with circadian biomarkers: a prospective and versatile tool for crime scene reconstruction

Linking biological samples found at a crime scene with the actual crime event represents the most important aspect of forensic investigation, together with the identification of the sample donor. While DNA profiling is well established for donor identification, no reliable methods exist for timing forensic samples. Here, we provide for the first time a biochemical approach for determining deposition time of human traces. Using commercial enzyme-linked immunosorbent assays we showed that the characteristic 24-h profiles of two circadian hormones, melatonin (concentration peak at late night) and cortisol (peak in the morning) can be reproduced from small samples of whole blood and saliva. We further demonstrated by analyzing small stains dried and stored up to 4 weeks the in vitro stability of melatonin, whereas for cortisol a statistically significant decay with storage time was observed, although the hormone was still reliably detectable in 4-week-old samples. Finally, we showed that the total protein concentration, also assessed using a commercial assay, can be used for normalization of hormone signals in blood, but less so in saliva. Our data thus demonstrate that estimating normalized concentrations of melatonin and cortisol represents a prospective approach for determining deposition time of biological trace samples, at least from blood, with promising expectations for forensic applications. In the broader context, our study opens up a new field of circadian biomarkers for deposition timing of forensic traces; future studies using other circadian biomarkers may reveal if the time range offered by the two hormones studied here can be specified more exactly

Cell Death by SecTRAPs: Thioredoxin Reductase as a Prooxidant Killer of Cells

BACKGROUND: SecTRAPs (selenium compromised thioredoxin reductase-derived apoptotic proteins) can be formed from the selenoprotein thioredoxin reductase (TrxR) by targeting of its selenocysteine (Sec) residue with electrophiles, or by its removal through C-terminal truncation. SecTRAPs are devoid of thioredoxin reductase activity but can induce rapid cell death in cultured cancer cell lines by a gain of function. PRINCIPAL FINDINGS: Both human and rat SecTRAPs killed human A549 and HeLa cells. The cell death displayed both apoptotic and necrotic features. It did not require novel protein synthesis nor did it show extensive nuclear fragmentation, but it was attenuated by use of caspase inhibitors. The redox active disulfide/dithiol motif in the N-terminal domain of TrxR had to be maintained for manifestation of SecTRAP cytotoxicity. Stopped-flow kinetics showed that NADPH can reduce the FAD moiety in SecTRAPs at similar rates as in native TrxR and purified SecTRAPs could maintain NADPH oxidase activity, which was accelerated by low molecular weight substrates such as juglone. In a cellular context, SecTRAPs triggered extensive formation of reactive oxygen species (ROS) and consequently antioxidants could protect against the cell killing by SecTRAPs. CONCLUSIONS: We conclude that formation of SecTRAPs could contribute to the cytotoxicity seen upon exposure of cells to electrophilic agents targeting TrxR. SecTRAPs are prooxidant killers of cells, triggering mechanisms beyond those of a mere loss of thioredoxin reductase activity