Positive and negative regulation of cytokines such as IFN-γ are key to normal homeostatic function. Negative regulation of IFN-γ in cells occurs via proteins called suppressors of cytokine signaling (SOCS)1 and -3. SOCS-1 inhibits IFN-γ function by binding to the autophosphorylation site of the tyrosine kinase Janus kinase (JAK)2. We have developed a short 12-mer peptide, WLVFFVIFYFFR, that binds to the autophosphorylation site of JAK2, resulting in inhibition of its autophosphorylation as well as its phosphorylation of IFN-γ receptor subunit IFNGR-1. The JAK2 tyrosine kinase inhibitor peptide (Tkip) did not bind to or inhibit tyrosine autophosphorylation of vascular endothelial growth factor receptor or phosphorylation of a substrate peptide by the protooncogene tyrosine kinase c-src. Tkip also inhibited epidermal growth factor receptor autophosphorylation, consistent with the fact that epidermal growth factor receptor is regulated by SOCS-1 and SOCS-3, similar to JAK2. Although Tkip binds to unphosphorylated JAK2 autophosphorylation site peptide, it binds significantly better to tyrosine-1007 phosphorylated JAK2 autophosphorylation site peptide. SOCS-1 only recognizes the JAK2 site in its phosphorylated state. Thus, Tkip recognizes the JAK2 autophosphorylation site similar to SOCS-1, but not precisely the same way. Consistent with inhibition of JAK2, Tkip inhibited the ability of IFN-γ to induce an antiviral state as well as up-regulate MHC class I molecules on cells at a concentration of ∼10 μM. This is similar to the Kd of SOCS-3 for the erythropoietin receptor. These data represent a proof-of-concept demonstration of a peptide mimetic of SOCS-1 that regulates JAK2 tyrosine kinase function.
Environmental DNA (eDNA) is a highly sensitive and cost‐effective tool that is increasingly being applied to studies of biodiversity and species detection. This non‐invasive method relies on the collection of environmental samples that contain genetic material being shed into surrounding environment by the target organism/s. While forensic science has a long history of using molecular tools for collecting DNA from the environment, the detection of human DNA from environmental water samples has been limited. This study investigated the detection and degradation rates of human eDNA in water samples under controlled laboratory conditions. Using a human‐specific qPCR assay targeting the ND1 region of human mitochondrial DNA, eDNA degradation over time in water spiked with human blood was assessed. Recovery of nuclear DNA was investigated by determining if routine DNA short tandem repeat (STR) profiles of the blood source could be generated. Results demonstrated that human eDNA remains detectable for up to 11 days under laboratory conditions in environmental water and up to 35 days in distilled water. Partial STR profiles could be recovered from environmental water only up to 24 h, while, in distilled water, partial profiles continued to be recovered up to 840 h. These findings demonstrate that sampling human eDNA from aquatic samples can provide reliable human DNA detection within relatively short time windows, assisting law enforcement agencies by providing information about the potential time an individual may have been present in an area or assisting in the detection and location of a body or remains in aquatic environments.
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