An approach to zwitterionic peptide design for colorimetric detection of the Southampton norovirus SV3CP protease.

Noroviruses are highly contagious and are one of the leading causes of acute gastroenteritis worldwide. Due to a lack of effective antiviral therapies, there is a need to diagnose and surveil norovirus infections to implement quarantine protocols and prevent large outbreaks. Currently, the gold standard of diagnosis uses reverse transcription polymerase chain reaction (RT-PCR), but PCR can have limited availability. Here, we propose a combination of a tunable peptide substrate and gold nanoparticles (AuNPs) to colorimetrically detect the Southampton norovirus 3C-like protease (SV3CP), a key protease in viral replication. Careful design of the substrate employs a zwitterionic peptide with opposite charged moieties on the C- and N- termini to induce a rapid color change visible to the naked eye; thus, this color change is indicative of SV3CP activity. This work expands on existing zwitterionic peptide strategies for protease detection by systematically evaluating the effects of lysine and arginine on nanoparticle charge screening. We also determine a limit of detection for SV3CP of 28.0 nM with comparable results in external breath condensate, urine, and fecal matter for 100 nM of SV3CP. The key advantage of this system is its simplicity and accessibility, thus making it an attractive tool for qualitative point-of-care diagnostics.

Regeneration of hyaline-like cartilage in situ with SOX9 stimulation of bone marrow-derived mesenchymal stem cells.

Microfracture, a common procedure for treatment of cartilage injury, induces fibrocartilage repair by recruiting bone marrow derived mesenchymal stem cells (MSC) to the site of cartilage injury. However, fibrocartilage is inferior biomechanically to hyaline cartilage. SRY-type high-mobility group box-9 (SOX9) is a master regulator of chondrogenesis by promoting proliferation and differentiation of MSC into chondrocytes. In this study we aimed to test the therapeutic potential of cell penetrating recombinant SOX9 protein in regeneration of hyaline cartilage in situ at the site of cartilage injury. We generated a recombinant SOX9 protein which was fused with super positively charged green fluorescence protein (GFP) (scSOX9) to facilitate cell penetration. scSOX9 was able to induce chondrogenesis of bone marrow derived MSC in vitro. In a rabbit cartilage injury model, scSOX9 in combination with microfracture significantly improved quality of repaired cartilage as shown by macroscopic appearance. Histological analysis revealed that the reparative tissue induced by microfracture with scSOX9 had features of hyaline cartilage; and collagen type II to type I ratio was similar to that in normal cartilage. This short term in vivo study demonstrated that when administered at the site of microfracture, scSOX9 was able to induce reparative tissue with features of hyaline cartilage.