David G. Gorenstein

Improving vascular maturation using noncoding RNAs increases antitumor effect of chemotherapy

Current antiangiogenesis therapy relies on inhibiting newly developed immature tumor blood vessels and starving tumor cells. This strategy has shown transient and modest efficacy. Here, we report a better approach to target cancer-associated endothelial cells (ECs), reverse permeability and leakiness of tumor blood vessels, and improve delivery of chemotherapeutic agents to the tumor. First, we identified deregulated microRNAs (miRs) from patient-derived cancer-associated ECs. Silencing these miRs led to decreased vascular permeability and increased maturation of blood vessels. Next, we screened a thioaptamer (TA) library to identify TAs selective for tumor-associated ECs. An annexin A2-targeted TA was identified and used for delivery of miR106b-5p and miR30c-5p inhibitors, resulting in vascular maturation and antitumor effects without inducing hypoxia. These findings could have implications for improving vascular-targeted therapy.

Acquisition of data at multiple gains within a single thermal melt experiment using the Rotor-Gene Q instrument

BackgroundRotor-Gene Q instrument was used to perform high-resolution protein thermal melt studies to characterize protein-small-molecule interaction. Fluorescent dye (1-anilino-8-naphthalenesulfonate (1,8-ANS)) is used as a reporter of protein unfolding to measure the protein melting temperature (Tm). Variations in the fluorescence yield upon titration of small molecules with the protein resulted in poor melting curves at low gain while a high gain setting caused signal saturation leading to data loss.FindingsAcquisition of data at multiple gains within a single experiment provided high-quality data for samples with both low and high fluorescence yields. The melting temperatures were measured for all the samples in one run, while avoiding loss of data due to signal saturation. This method was successfully used to measure the binding constant by titration of a small-molecule ligand with the target protein.ConclusionProtein thermal melt experiments using the Rotor-Gene Q instrument have been made feasible for samples that show variations in fluorescence yield. Furthermore, since protein melting is irreversible, using multiple gains in the same experiment prevented loss of sample and saved gain optimization time.