DNA-Barcoded Plasmonic Nanostructures for Activity-Based Protease Sensing.

We report the development of a new class of protease activity sensors called DNA-barcoded plasmonic nanostructures. These probes are comprised of gold nanoparticles functionalized with peptide-DNA conjugates (GPDs), where the peptide is a substrate of the protease of interest. The DNA acts as a barcode identifying the peptide and facilitates signal amplification. Protease-mediated peptide cleavage frees the DNA from the nanoparticle surface, which is subsequently measured via a CRISPR/Cas12a-based assay as a proxy for protease activity. As proof-of-concept, we show activity-based, multiplexed detection of the SARS-CoV-2-associated protease, 3CL, and the apoptosis marker, caspase 3, with high sensitivity and selectivity. GPDs yield >25-fold turn-on signals, 100-fold improved response compared to commercial probes, and detection limits as low as 58 pM at room temperature. Moreover, nanomolar concentrations of proteases can be detected visually by leveraging the aggregation-dependent color change of the gold nanoparticles. We showcase the clinical potential of GPDs by detecting a colorectal cancer-associated protease, cathepsin B, in three different patient-derived cell lines. Taken together, GPDs detect physiologically relevant concentrations of active proteases in challenging biological samples, require minimal sample processing, and offer unmatched multiplexing capabilities (mediated by DNA), making them powerful chemical tools for biosensing and disease diagnostics.

Elucidating the direct effects of the novel HDAC inhibitor bocodepsin (OKI-179) on T cells to rationally design regimens for combining with immunotherapy.

Histone deacetylase inhibitors (HDACi) are currently being explored for the treatment of both solid and hematological malignancies. Although originally thought to exert cytotoxic responses through tumor-intrinsic mechanisms by increasing expression of tumor suppressor genes, several studies have demonstrated that therapeutic responses depend on an intact adaptive immune system: particularly CD8 T cells. It is therefore critical to understand how HDACi directly affects T cells in order to rationally design regimens for combining with immunotherapy. In this study, we evaluated T cell responses to a novel class-selective HDACi (OKI-179, bocodepsin) by assessing histone acetylation levels, which revealed rapid responsiveness accompanied by an increase in CD4 and CD8 T cell frequencies in the blood. However, these rapid responses were transient, as histone acetylation and frequencies waned within 24 hours. This contrasts with in vitro models where high acetylation was sustained and continuous exposure to HDACi suppressed cytokine production. In vivo comparisons demonstrated that stopping OKI-179 treatment during PD-1 blockade was superior to continuous treatment. These findings provide novel insight into the direct effects of HDAC inhibitors on T cells and that treatment schedules that take into account acute T cell effects should be considered when combined with immunotherapies in order to fully harness the tumor-specific T cell responses in patients.

Vitamin D binding protein greatly improves bioactivity but is not essential for orally administered vitamin D.

Vitamin D3 is a prohormone that is essential for calcium homeostasis. It is naturally produced in the skin by ultraviolet-B (UVB) irradiation of 7-dehydrocholesterol. In the absence of skin production, vitamin D3 can also be obtained from oral sources. However, the actual biological equivalence of naturally produced (i.e., UVB-irradiation of skin) and oral vitamin D3  has not been determined. We previously identified a unique and specific transport mechanism for skin-generated vitamin D3 which requires vitamin D binding protein (DBP); a mechanism that differs from absorption and transport of oral vitamin D3 . In the following report, we examined the impact of this difference on the biological activity of vitamin D3 . We report that UVB-generated vitamin D3 is more potent at raising serum calcium compared to oral vitamin D3 , with the total biological activity being twofold higher. By examining the excretion of radiolabeled vitamin D3 injected unbound or pre-bound by DBP, we attributed the increased activity of skin-generated vitamin D3 to a significant reduction in biliary excretion of DBP-bound vitamin D relative to unbound vitamin D. Thus, removal of vitamin D3 from the skin by the natural DBP system markedly improves biological activity compared to that given orally.