Assessing the use of environmental DNA (eDNA) as a tool in the detection of human DNA in water.

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.

Detecting marine pests using environmental DNA and biophysical models.

The spread of marine pests is occurring at record rates due to globalisation and increasing trade. Environmental DNA (eDNA) is an emerging tool for pest surveillance, allowing for the detection of genetic material shed by organisms into the environment. However, factors influencing the spatial and temporal detection limits of eDNA in marine environments are poorly understood. In this study we use eDNA assays to assess the invasive ranges of two marine pests in south-eastern Australia, the kelp Undaria pinnatifida and the seastar Asterias amurensis. We explored the temporal and spatial detection limits of eDNA under different oceanographic conditions by combining estimates of eDNA decay with biophysical modelling. Positive eDNA detections at several new locations indicate the invasive range of both pest species is likely to be wider than currently assumed. Environmental DNA decay rates were similar for both species, with a decay rate constant of 0.035 h-1 for U. pinnatifida, and a decay rate constant of 0.041 h-1 for A. amurensis, resulting in a 57-73% decrease in eDNA concentrations in the first 24 h and decaying beyond the limits of detection after 3-4 days. Biophysical models informed by eDNA decay profiles indicate passive transport of eDNA up to a maximum of 10 to 20 km from its source, with a ~90-95% reduction in eDNA concentration within 1-3 km from the source, depending on local oceanography. These models suggest eDNA signals are likely to be highly localised, even in complex marine environments. This was confirmed with spatially replicated eDNA sampling around an established U. pinnatifida population indicating detection limits of ~750 m from the source. This study highlights the value of eDNA methods for marine pest surveillance and provides a much-needed description of the spatio-temporal detection limits of eDNA under different oceanographic conditions.

Characterization of a peptide inhibitor of Janus kinase 2 that mimics suppressor of cytokine signaling 1 function.

Positive and negative regulation of cytokines such as IFN-gamma are key to normal homeostatic function. Negative regulation of IFN-gamma in cells occurs via proteins called suppressors of cytokine signaling (SOCS)1 and -3. SOCS-1 inhibits IFN-gamma 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-gamma 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-gamma to induce an antiviral state as well as up-regulate MHC class I molecules on cells at a concentration of approximately 10 microM. This is similar to the K(d) 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.