A Dual Promoter System to Monitor IFN-γ Signaling in vivo at Single-cell Resolution.

IFN-γ secreted from immune cells exerts pleiotropic effects on tumor cells, including induction of immune checkpoint and antigen presentation, growth inhibition, and apoptosis induction. We combined a dual promoter system with an IFN-γ signaling responsive promoter to generate a reporter named the interferon sensing probe (ISP), which quantitates the response to IFN-γ by means of fluorescence and bioluminescence. The integration site effect of the transgene is compensated for by the PGK promoter-driven expression of a fluorescent protein. Among five potential IFN-γ-responsive elements, we found that the interferon γ-activated sequence (GAS) exhibited the best performance. When ISP-GAS was introduced into four cell lines and subjected to IFN-γ stimulation, dose-dependency was observed with an EC50 ranging from 0.2 to 0.9 ng/mL, indicating that ISP-GAS can be generally used as a sensitive biosensor of IFN-γ response. In a syngeneic transplantation model, the ISP-GAS-expressing cancer cells exhibited bioluminescence and fluorescence signals in an IFN-γ receptor-dependent manner. Thus, ISP-GAS could be used to quantitatively monitor the IFN-γ response both in vitro and in vivo.Key words: in vivo imaging, tumor microenvironment, interferon-gamma, dual promoter system.

Evaluation of Amyloid Polypeptide Aggregation Inhibition and Disaggregation Activity of A-Type Procyanidins.

The number of people worldwide suffering from Alzheimer's disease (AD) and type 2 diabetes (T2D) is on the rise. Amyloid polypeptides are thought to be associated with the onset of both diseases. Amyloid-ß (Aß) that aggregates in the brain and human islet amyloid polypeptide (hIAPP) that aggregates in the pancreas are considered cytotoxic and the cause of the development of AD and T2D, respectively. Thus, inhibiting amyloid polypeptide aggregation and disaggregation existing amyloid aggregates are promising approaches in the therapy and prevention against both diseases. Therefore, in this research, we evaluated the Aß/hIAPP anti-aggregation and disaggregation activities of A-type procyanidins 1-7 and their substructures 8 and 9, by conducting structure-activity relationship studies and identified the active site. The thioflavin-T (Th-T) assay, which quantifies the degree of aggregation of amyloid polypeptides based on fluorescence intensity, and transmission electron microscopy (TEM), employed to directly observe amyloid polypeptides, were used to evaluate the activity. The results showed that catechol-containing compounds 1-6 exhibited Aß/hIAPP anti-aggregation and disaggregation activities, while compound 7, without catechol, showed no activity. This suggests that the presence of catechol is important for both activities. Daily intake of foods containing A-type procyanidins may be effective in the prevention and treatment of both diseases.