One-Step Preservation of Phosphoproteins and Tissue Morphology at Room Temperature for Diagnostic and Research Specimens

BACKGROUND: There is an urgent need to measure phosphorylated cell signaling proteins in cancer tissue for the individualization of molecular targeted kinase inhibitor therapy. However, phosphoproteins fluctuate rapidly following tissue procurement. Snap-freezing preserves phosphoproteins, but is unavailable in most clinics and compromises diagnostic morphology. Formalin fixation preserves tissue histomorphology, but penetrates tissue slowly, and is unsuitable for stabilizing phosphoproteins. We originated and evaluated a novel one-step biomarker and histology preservative (BHP) chemistry that stabilizes signaling protein phosphorylation and retains formalin-like tissue histomorphology with equivalent immunohistochemistry in a single paraffin block. RESULTS: Total protein yield extracted from BHP-fixed, routine paraffin-embedded mouse liver was 100% compared to snap-frozen tissue. The abundance of 14 phosphorylated proteins was found to be stable over extended fixation times in BHP fixed paraffin embedded human colon mucosa. Compared to matched snap-frozen tissue, 8 phosphoproteins were equally preserved in mouse liver, while AMPKβ1 Ser108 was slightly elevated after BHP fixation. More than 25 tissues from mouse, cat and human specimens were evaluated for preservation of histomorphology. Selected tissues were evaluated in a multi-site, independent pathology review. Tissue fixed with BHP showed equivalent preservation of cytoplasmic and membrane cytomorphology, with significantly better nuclear chromatin preservation by BHP compared to formalin. Immunohistochemical staining of 13 non-phosphorylated proteins, including estrogen receptor alpha, progesterone receptor, Ki-67 and Her2, was equal to or stronger in BHP compared to formalin. BHP demonstrated significantly improved immunohistochemical detection of phosphorylated proteins ERK Thr202/Tyr204, GSK3-α/β Ser21/Ser9, p38-MAPK Thr180/Tyr182, eIF4G Ser1108 and Acetyl-CoA Carboxylase Ser79. CONCLUSION: In a single paraffin block BHP preserved the phosphorylation state of several signaling proteins at a level comparable to snap-freezing, while maintaining the full diagnostic immunohistochemical and histomorphologic detail of formalin fixation. This new tissue fixative has the potential to greatly facilitate personalized medicine, biobanking, and phospho-proteomic research

Oral administration of oleic or linoleic acid accelerates the inflammatory phase of wound healing

The effects of oral ingestion of oleic (OLA) and linoleic (LNA) acids on wound healing in rats were investigated. LNA increased the influx of inflammatory cells, the concentration of hydrogen peroxide (H2O2) and cytokine-induced neutrophil chemoattractant-2?? (CINC-2??), and the activation of the transcription factor activator protein-1 (AP-1) in the wound at 1?hour post wounding. LNA decreased the number of inflammatory cells and IL-1, IL-6, and macrophage inflammatory protein-3 (MIP-3) concentrations, as well as NF-?B activation in the wound at 24?hours post wounding. LNA accelerated wound closure over a period of 7 days. OLA increased TNF-? concentration and NF-?B activation at 1?hour post wounding. A reduction of IL-1, IL-6, and MIP-3? concentrations, as well as NF-?B activation, was observed 24?hours post wounding in the OLA group. These data suggest that OLA and LNA accelerate the inflammatory phase of wound healing, but that they achieve this through different mechanism

Estimating wound age: looking into the future

A critical review is made of the studies on wound healing used for forensic purposes, focusing on the problem of which characteristics indicate that a parameter could be used as evidence in court. A panel analysing the more important information obtained by each marker is given, and a perspective of what might be expected from future research is discussed