Detection limits are central to improve reporting standards when using Nile red for microplastic quantification.

Beyond simple identification of either the presence or absence of microplastic particles in the environment, quantitative accuracy has been criticised as being neither comparable nor reproducible. This is, in part, due to difficulties in the identification of synthetic particles amidst naturally occurring organic and inorganic components. The fluorescent stain Nile red has been proposed as a tool to overcome this issue, but to date, has been used without consideration of polymer specific fluorescent variability. The aim of this study was to evaluate the efficacy of Nile red for microplastic detection by systematically investigating what drives variations in particle pixel brightness (PPB). The results showed that PPB varied between polymer type, shape, size, colour and by staining procedure. Sand, an inorganic component of the sample matrix does not fluoresce when stained with Nile red. In contrast the organic components, wood and chitin, fluoresce between 1.40 and 12 arbitrary units (a.u.) and 32 and 74 a.u. after Nile red staining, respectively. These data informed the use of a PPB threshold limit of 100 a.u., which improved the detection of EPS, HDPE, PP and PA-6 from the 6 polymers tested and reduced analysis time by 30-58% compared to unstained samples. Conversely, as with traditional illumination, PET and PVC were not accurately estimated using this approach. This study shows that picking a threshold limit is not arbitrary but rather must be informed by polymer specific fluorescent variability and matrix considerations. This is an essential step needed to facilitate comparability and reproducibility between individual studies.

Downmodulation of Effector Functions in NK Cells upon Toxoplasma gondii Infection.

The obligate intracellular parasite Toxoplasma gondii can actively infect any nucleated cell type, including cells from the immune system. The rapid transfer of T. gondii from infected dendritic cells to effector natural killer (NK) cells may contribute to the parasite's sequestration and shielding from immune recognition shortly after infection. However, subversion of NK cell functions, such as cytotoxicity or production of proinflammatory cytokines, such as gamma interferon (IFN-γ), upon parasite infection might also be beneficial to the parasite. In the present study, we investigated the effects of T. gondii infection on NK cells. In vitro, infected NK cells were found to be poor at killing target cells and had reduced levels of IFN-γ production. This could be attributed in part to the inability of infected cells to form conjugates with their target cells. However, even upon NK1.1 cross-linking of NK cells, the infected NK cells also exhibited poor degranulation and IFN-γ production. Similarly, NK cells infected in vivo were also poor at killing target cells and producing IFN-γ. Increased levels of transforming growth factor ß production, as well as increased levels of expression of SHP-1 in the cytosol of infected NK cells upon infection, were observed in infected NK cells. However, the phosphorylation of STAT4 was not altered in infected NK cells, suggesting that transcriptional regulation mediates the reduced IFN-γ production, which was confirmed by quantitative PCR. These data suggest that infection of NK cells by T. gondii impairs NK cell recognition of target cells and cytokine release, two mechanisms that independently could enhance T. gondii survival.