New Methods for the Quantification of Ingested Nano- and Ultrafine Plastics in Seabirds.

Plastic ingestion has been documented in a plethora of taxa. However, there is a significant gap in the detection of nano- and ultrafine particles due to size limitations of commonly used techniques. Using two Australian seabird species as case studies, the flesh-footed shearwater (FFSH) Ardenna carneipes and short-tailed shearwater (STSH) A. tenuirostris, we tested a novel approach of flow cytometry to quantify ingested particles <70 µm in the fecal precursor (guano; colon and cloacal contents) of both species. This method provided the first baseline data set for these species for plastics in the 200 nm-70 µm particle size ranges and detected a mean of 553.50 ± 91.21 and 350.70 ± 52.08 plastics (count/mg fecal precursor, wet mass) in STSH and FFSH, respectively, whereas Fourier transform infrared spectroscopy (FT-IR) provided accurate measurements of polymer compositions and quantities in the size range above 5.5 × 5.5 µm2. The abundance of nano- and ultrafine particles in the guano (count/mg) was not significantly different between species (p-value = 0.051), suggesting that foraging distribution or prey items, but not species, may contribute to the consumption of small plastics. In addition, there was no correlation between macroplastics in the stomach compared to the fecal precursor, indicating that small particles are likely bioaccumulating (e.g., through shedding and digestive fragmentation) and/or being directly ingested. Combining flow cytometry with FT-IR provides a powerful quantitative and qualitative analysis tool for detecting particles orders of magnitude smaller than that are currently explored with wider applications across taxa and marine environments.

Novel Methods to Manipulate Autolysis in Sparkling Wine: Effects on Yeast.

Sparkling wine made by the traditional method (Méthode Traditionelle) develops a distinct and desirable flavour and aroma profile attributed to proteolytic processes during prolonged ageing on lees. Microwave, ultrasound and addition of ß-glucanase enzymes were applied to accelerate the disruption of Saccharomyces cerevisiae, and added to the tirage solution for secondary fermentation in traditional sparkling winemaking. Scanning electron microscopy and flow cytometry analyses were used to observe and describe yeast whole-cell anatomy, and cell integrity and structure via propidium iodide (PI) permeability after 6-, 12- and 18-months post-tirage. Treatments applied produced features on lees that were distinct from that of the untreated control yeast. Whilst control yeast displayed budding cells (growth features) with smooth, cavitated and flat external cell appearances; microwave treated yeast cells exhibited modifications like 'doughnut' shapes immediately after treatment (time 0). Similar 'doughnut'-shaped and 'pitted/porous' cell features were observed on progressively older lees from the control. Flow cytometry was used to discriminate yeast populations; features consistent with cell disruption were observed in the microwave, ultrasound and enzyme treatments, as evidenced by up to 4-fold increase in PI signal in the microwave treatment. Forward and side scatter signals reflected changes in size and structure of yeast cells, in all treatments applied. When flow cytometry was interpreted alongside the scanning electron microscopy images, bimodal populations of yeast cells with low and high PI intensities were revealed and distinctive 'doughnut'-shaped cell features observed in association with the microwave treatment only at tirage, that were not observed until 12 months wine ageing in older lees from the control. This work offers both a rapid approach to visualise alterations to yeast cell surfaces and a better understanding of the mechanisms of yeast lysis. Microwave, ultrasound or ß-glucanase enzymes are tools that could potentially initiate the release of yeast cell compounds into wine. Further investigation into the impact of such treatments on the flavour and aroma profiles of the wines through sensory evaluation is warranted.

Tasmanian devil CD28 and CTLA4 capture CD80 and CD86 from adjacent cells.

Immune checkpoint immunotherapy is a pillar of human oncology treatment with potential for non-human species. The first checkpoint immunotherapy approved for human cancers targeted the CTLA4 protein. CTLA4 can inhibit T cell activation by capturing and internalizing CD80 and CD86 from antigen presenting cells, a process called trans-endocytosis. Similarly, CD28 can capture CD80 and CD86 via trogocytosis and retain the captured ligands on the surface of the CD28-expressing cells. The wild Tasmanian devil (Sarcophilus harrisii) population has declined by 77% due to transmissible cancers that evade immune defenses despite genetic mismatches between the host and tumors. We used a live cell-based assay to demonstrate that devil CTLA4 and CD28 can capture CD80 and CD86. Mutation of evolutionarily conserved motifs in CTLA4 altered functional interactions with CD80 and CD86 in accordance with patterns observed in other species. These results suggest that checkpoint immunotherapies can be translated to evolutionarily divergent species.

Generation and Testing of Fluorescent Adaptable Simple Theranostic (FAST) Proteins.

This protocol provides a step-by-step method to create recombinant fluorescent fusion proteins that can be secreted from mammalian cell lines. This builds on many other recombinant protein and fluorescent protein techniques, but is among the first to harness fluorescent fusion proteins secreted directly into cell culture supernatant. This opens new possibilities that are not achievable with proteins produced in bacteria or yeast, such as direct use of the fluorescent protein-secreting cells in live co-culture assays. The Fluorescent Adaptable Simple Theranostic (FAST) protein system includes a histidine purification tag and a tobacco etch virus (TEV) cleavage site, allowing the purification tag and fluorescent protein to be removed for therapeutic use. This protocol is split into five parts: (A) In silico characterization of the gene-of-interest (GOI) and protein-of-interest (POI); (B) design of the expression vector; (C) assembly of the expression vector; (D) transfection of a eukaryotic cell line with the expression vector; (E) testing of the recombinant protein. This extensive protocol can be completed with only polymerase chain reaction (PCR) and cell culture training. Additionally, each part of the protocol can be used independently.

A novel system to map protein interactions reveals evolutionarily conserved immune evasion pathways on transmissible cancers.

Around 40% of humans and Tasmanian devils (Sarcophilus harrisii) develop cancer in their lifetime, compared to less than 10% for most species. In addition, devils are affected by two of the three known transmissible cancers in mammals. Immune checkpoint immunotherapy has transformed human medicine, but a lack of species-specific reagents has limited checkpoint immunology in most species. We developed a cut-and-paste reagent development system and used the fluorescent fusion protein system to show that immune checkpoint interactions are conserved across 160,000,000 years of evolution, CD200 is highly expressed on transmissible tumor cells, and coexpression of CD200R1 can block CD200 surface expression. The system's versatility across species was demonstrated by fusing a fluorescent reporter to a camelid-derived nanobody that binds human programmed death ligand 1. The evolutionarily conserved pathways suggest that naturally occurring cancers in devils and other species can be used to advance our understanding of cancer and immunological tolerance.

Mouse Model of Devil Facial Tumour Disease Establishes That an Effective Immune Response Can be Generated Against the Cancer Cells.

The largest carnivorous marsupial in Australia, the Tasmanian devil (Sarcophilus harrisii) is facing extinction in the wild due to a transmissible cancer known as Devil Facial Tumour Disease (DFTD). DFTD is a clonal cell line transmitted from host to host with 100% mortality and no known immunity. While it was first considered that low genetic diversity of the population of devils enabled the allograft transmission of DFTD recent evidence reveals that genetically diverse animals succumb to the disease. The lack of an immune response against the DFTD tumor cells may be due to a lack of immunogenicity of the tumor cells. This could facilitate transmission between devils. To test immunogenicity, mice were injected with viable DFTD cells and anti-DFTD immune responses analyzed. A range of antibody isotypes against DFTD cells was detected, indicating that as DFTD cells can induce an immune response they are immunogenic. This was supported by cytokine production, when splenocytes from mice injected with DFTD cells were cultured in vitro with DFTD cells and the supernatant analyzed. There was a significant production of IFN-γ and TNF-α following the first injection with DFTD cells and a significant production of IL-6 and IL-10 following the second injection. Splenocytes from naïve or immunized mice killed DFTD cells in in vitro cytotoxicity assays. Thus, they are also targets for immunological destruction. We conclude that as an immune response can be generated against DFTD cells they would be suitable targets for a vaccine.