The Wnts are secreted, lipidated glycoproteins that play a role in cellular processes of differentiation, proliferation, migration, survival, polarity and stem cell self-renewal. The majority of Wnts biological effects are through binding to specific frizzled (Fzd) receptor complexes leading to activation of downstream pathways. Secreted frizzled-related proteins (sFRPs) were first identified as antagonists of Wnt signalling by binding directly to Wnts. They comprise two domains, a Fzd-like cysteine rich domain (CRD) and a netrin-like domain (NLD). Subsequently sFRPs have been shown to also interact with Fzd receptors and more diverse functions have been identified, including potentiation of Wnt signalling. Many aspects of the biology of this family remain to be elucidated. We used the number and brightness (N&B) method, a technique based on fluorescence fluctuation analysis, to characterise the intracellular aggregation and trafficking of sFRP4 domains. We expressed sFRP4 and its' domains as EGFP fusions and then characterised the effect of endogenous Wnt3a by fluorescence confocal imaging. We observed vesicular trafficking of sFRP4 and that the NLD domain has a vesicular association signal. We found that sFRP4 and the CRD formed oligomeric aggregates in the perinuclear region while the NLD was distributed evenly throughout the cell with a larger proportion of aggregates. Most significantly we observed intracellular redistribution of sFRP4 in response to Wnt3a suggesting that Wnt3a can modulate intracellular localisation and secretion of sFRP4. Our results reveal a number of novel findings regarding sFRP4 which are likely to have relevance to this wider family.

Profilin, a small cytoskeletal protein, and phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] have been implicated in cellular events that alter the cell morphology, such as endocytosis, cell motility, and formation of the cleavage furrow during cytokinesis. Profilin has been shown to interact with PI(4,5)P2, but the role of this interaction is still poorly understood. Using giant unilamellar vesicles (GUVs) as a simple model of the cell membrane, we investigated the interaction between profilin and PI(4,5)P2. A number and brightness analysis demonstrated that in the absence of profilin, molar ratios of PI(4,5)P2 above 4% result in lipid demixing and cluster formations. Furthermore, adding profilin to GUVs made with 1% PI(4,5)P2 leads to the formation of clusters of both profilin and PI(4,5)P2. However, due to the self-quenching of the dipyrrometheneboron difluoride-labeled PI(4,5)P2, we were unable to determine the size of these clusters. Finally, we show that the formation of these clusters results in the destabilization and deformation of the GUV membrane.

Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), is one of the major environmental pollutants that causes mutagenesis and cancer. BaP has been shown to accumulate in phytoplankton and zooplankton. We have studied the localization and aggregation of BaP in Chlorella sp., a microalga that is one of the primary producers in the food chain, using fluorescence confocal microscopy and fluorescence lifetime imaging microscopy with the phasor approach to characterize the location and the aggregation of BaP in the cell. Our results show that BaP accumulates in the lipid bodies of Chlorella sp. and that there is Förster resonance energy transfer between BaP and photosystems of Chlorella sp., indicating the close proximity of the two molecular systems. The lifetime of BaP fluorescence was measured to be 14 ns in N,N-dimethylformamide, an average of 7 ns in Bold's basal medium, and 8 ns in Chlorella cells. Number and brightness analysis suggests that BaP does not aggregate inside Chlorella sp. (average brightness = 5.330), while it aggregates in the supernatant. In Chlorella grown in sediments spiked with BaP, in 12 h the BaP uptake could be visualized using fluorescence microscopy.

Gastrointestinal (GI) bleeding causes more than 300 000 hospitalizations per year in the United States. Imaging plays a crucial role in accurately locating the source of the bleed for timely intervention. Magnetic particle imaging (MPI) is an emerging clinically translatable imaging modality that images superparamagnetic iron-oxide (SPIO) tracers with extraordinary contrast and sensitivity. This linearly quantitative modality has zero background tissue signal and zero signal depth attenuation. MPI is also safe: there is zero ionizing radiation exposure to the patient and clinically approved tracers can be used with MPI. In this study, we demonstrate the use of MPI along with long-circulating, PEG-stabilized SPIOs for rapid in vivo detection and quantification of GI bleed. A mouse model genetically predisposed to GI polyp development (ApcMin/+) was used for this study, and heparin was used as an anticoagulant to induce acute GI bleeding. We then injected MPI-tailored, long-circulating SPIOs through the tail vein, and tracked the tracer biodistribution over time using our custom-built high resolution field-free line (FFL) MPI scanner. Dynamic MPI projection images captured tracer accumulation in the lower GI tract with excellent contrast. Quantitative analysis of the MPI images show that the mice experienced GI bleed rates between 1 and 5 μL/min. Although there are currently no human scale MPI systems, and MPI-tailored SPIOs need to undergo further development and evaluation, clinical translation of the technique is achievable. The robust contrast, sensitivity, safety, ability to image anywhere in the body, along with long-circulating SPIOs lends MPI outstanding promise as a clinical diagnostic tool for GI bleeding.

Emergency room visits due to traumatic brain injury (TBI) is common, but classifying the severity of the injury remains an open challenge. Some subjective methods such as the Glasgow Coma Scale attempt to classify traumatic brain injuries, as well as some imaging based modalities such as computed tomography and magnetic resonance imaging. However, to date it is still difficult to detect and monitor mild to moderate injuries. In this report, we demonstrate that the magnetic particle imaging (MPI) modality can be applied to imaging TBI events with excellent contrast. MPI can monitor injected iron nanoparticles over long time scales without signal loss, allowing researchers and clinicians to monitor the change in blood pools as the wound heals.