Parallel feedback loops control the basal activity of the HOG MAPK signaling cascade.

Tight regulation of the MAP kinase Hog1 is crucial for survival under changing osmotic conditions. Interestingly, we found that Hog1 phosphorylates multiple upstream components, implying feedback regulation within the signaling cascade. Taking advantage of an unexpected link between glucose availability and Hog1 activity, we used quantitative single cell measurements and computational modeling to unravel feedback regulation operating in addition to the well-known adaptation feedback triggered by glycerol accumulation. Indeed, we found that Hog1 phosphorylates its activating kinase Ssk2 on several sites, and cells expressing a non-phosphorylatable Ssk2 mutant are partially defective for feedback regulation and proper control of basal Hog1 activity. Together, our data suggest that Hog1 activity is controlled by intertwined regulatory mechanisms operating with varying kinetics, which together tune the Hog1 response to balance basal Hog1 activity and its steady-state level after adaptation to high osmolarity.

Biological variation of established and novel biomarkers for atherosclerosis: Results from a prospective, parallel-group cohort study.

BACKGROUND: Biomarkers are a promising tool for the management of patients with atherosclerosis, but their variation is largely unknown. We assessed within-subject and between-subject biological variation of biomarkers in peripheral artery disease (PAD) patients and healthy controls, and defined which biomarkers have a favorable variation profile for future studies. METHODS: Prospective, parallel-group cohort study, including 62 patients with stable PAD (79% men, 65±7years) and 18 healthy control subjects (44% men, 57±7years). Blood samples were taken at baseline, and after 3-, 6-, and 12-months. We calculated within-subject (CVI) and between-subject (CVG) coefficients of variation and intra-class correlation coefficient (ICC). RESULTS: Mean levels of D-dimer, hs-CRP, IL-6, IL-8, MMP-9, MMP-3, S100A8/A9, PAI-1, sICAM-1, and sP-selectin levels were higher in PAD patients than in healthy controls (P≤.05 for all). CVI and CVG of the different biomarkers varied considerably in both groups. An ICC≥0.5 (indicating moderate-to-good reliability) was found for hs-CRP, D-Dimer, E-selectin, IL-10, MCP-1, MMP-3, oxLDL, sICAM-1 and sP-selectin in both groups, for sVCAM in healthy controls and for MMP-9, PAI-1 and sCD40L in PAD patients. CONCLUSIONS: Single biomarker measurements are of limited utility due to large within-subject variation, both in PAD patients and healthy subjects. D-dimer, hs-CRP, MMP-9, MMP-3, PAI-1, sP-selectin and sICAM-1 are biomarkers with both higher mean levels in PAD patients and a favorable variation profile making them most suitable for future studies.