Microplastics are globally recognized as contaminants in freshwater and marine aquatic systems. To date there is no universally accepted protocol for isolation and quantification of microplastics from aqueous media. Various methodologies exist, many of which are time consuming and have the potential to introduce contaminants into samples, thereby obscuring characterization of the environmental microplastic load. Here, we present first steps in the detection of microplastics in liquid samples, based on their fluorescent staining followed by high throughput analysis and quantification using Flow Cytometry. Using controlled laboratory settings nine polymer types [polystyrene (PS); polyethylene (PE); polyethylene terephthalate (PET/PETE); high density polyethylene (HDPE); low density polyethylene (LDPE); polyvinyl chloride (PVC); polypropylene (PP); nylon (PA); polycarbonate (PC)] were tested for identification and quantification in freshwater. All nine plastic types were stained with 10 µg/mL Nile Red in 10% dimethyl sulfoxide with a 10 min incubation time. The lowest spatial detectable limit for plastic particles was 200 nm. Out of the nine polymer types chosen for the study PS, PE, PET, and PC were well-identified; however, results for other plastic types (PVC, PP, PA, LDPE, and HDPE) were masked to certain extent by Nile Red aggregation and precipitation. The methodology presented here permits identification of a range of particle sizes and types. It represents a significant step in the quantification of microplastics by replacing visual data interpretation with a sensitive and automated method.

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Nuclear receptors are ligand-modulated transcription factors that transduce the presence of lipophilic ligands into changes in gene expression. Nuclear receptor activity is regulated by ligand-induced interactions with coactivator or corepressor molecules. From a positive hormone response element (pHRE) and in the absence of hormone, corepressors SMRT and N-CoR are bound to some nuclear receptors such as the thyroid hormone (T3Rs) and retinoic acid receptors and mediate inhibition of basal levels of transcription. Ligand binding results in dissociation of corepressors and association of coactivators, resulting in the reversal of inhibition and a net activation of transcription. However, the role of cofactors on the activity of nuclear receptors from negative HREs (nHREs) is poorly understood. Here we show that corepressor SMRT can act as a potent coactivator for T3R␣ from a nHRE; N-CoR has a similar but significantly attenuated activity. Mutagenesis of residues in the hinge region of T3R␣ that block binding of SMRT and N-CoR inhibits ligand-independent transcriptional activation by T3R␣ from a nHRE. These mutations also abrogate SMRT-mediated increase in transcriptional activity by T3R␣ at a nHRE without significantly affecting ligand-dependent activation at a pHRE. Partial protease digestion coupled to the mobility shift assay indicate differences in the conformation of T3R␣-SMRT complexes bound to a pHRE versus a nHRE. These results suggest that allosteric changes resulting from binding of T3R␣ to different response elements, i.e. pHREs versus nHREs, dictate whether a cofactor will function as a coactivator or a corepressor. This, in turn, greatly expands the repertoire of mechanisms used in modulating transcription without the need for expression of new regulatory molecules.Transcriptional regulation is fundamental to the normal functioning of the cell and is achieved through positively or negatively acting transcription factors (1, 2). Whereas in some cases the transcription factors that activate and repress gene expression are encoded by different genes, increasingly more transcription factors are now recognized to function both as an activator and a repressor depending on the nature of the response element that they interact with and the cellular context (1, 2). However, the details of how an activating transcription factor can become a repressor and vice versa is not well understood.One of the factors that can serve as a transcriptional activator or repressor depending on the response element and cellular context is the thyroid hormone receptor (T3R) 1 (3, 4), which belongs to the nuclear receptor superfamily of ligand modulated transcriptional factors (for reviews, see Refs. 5 and 6). Recent studies have begun to uncover the molecular details of this bimodal activity on a positive hormone response element (pHRE). In the absence of hormone, T3R associates with corepressor molecules, such as SMRT and N-CoR (7-9), which assemble a repressive complex that shuts down transcription (reviewed in Refs. 5 and 6)....

This study investigates different UV doses (mJ/cm(2)) and the effect of dark incubation on the survival of the algae Tetraselmis suecica, to simulate ballast water treatment and subsequent transport. Samples were UV irradiated and analyzed by flow cytometry and standard culturing methods. Doses of ≥400 mJ/cm(2) rendered inactivation after 1 day as measured by all analytical methods, and are recommended for ballast water treatment if immediate impairment is required. Irradiation with lower UV doses (100-200 mJ/cm(2)) gave considerable differences of inactivation between experiments and analytical methods. Nevertheless, inactivation increased with increasing doses and incubation time. We argue that UV doses ≥100 mJ/cm(2) and ≤200 mJ/cm(2) can be sufficient if the water is treated at intake and left in dark ballast tanks. The variable results demonstrate the challenge of giving unambiguous recommendations on duration of dark incubation needed for inactivation when algae are treated with low UV doses.

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