2 research outputs found
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BRET-based reporter conjugated luminescent nanoparticles for improved bioluminescence imaging
The emergence of bright bioluminescent luciferase reporters has enabled versatilebioimaging applications in vitro and in vivo . However, conventional luciferases such as
nanoluciferase are sub-optimal reporters for bioluminescence imaging (BLI) in vivo due to their
blue-shifted emission which is highly attenuated by tissue, resulting in a dim, less sensitive
signal. To overcome this limitation, Nluc and its substrate furimazine have been further
engineered to produce red-shifted emission. Additionally, Nluc has been fused to fluorescent
proteins, creating reporters with spectrally shifted light emission through bioluminescence
resonance energy transfer (BRET). In this study, we employed two BRET-based fusion reporters,
CeNLuc and LumiScarlet, as they have previously demonstrated improved imaging properties
compared to luciferase-only reporters. We aimed to immobilize CeNLuc and LumiScarlet onto
the E2 nanoparticle surface to develop brighter reporters for BLI. We successfully conjugated
CeNLuc and LumiScarlet onto E2 using the SpyCatcher-SpyTag conjugation, resulting in
CeNLuc-E2 and LumiScarlet-E2 nanoparticles of ~35-nm in size. Furthermore, we were able to
control the number of luminescent proteins per particle and demonstrated that CeNLuc-E2 and
LumiScarlet-E2 underwent efficient BRET. We also evaluated the luminescence of CeNLuc-E2
and LumiScarlet-E2 using BLI. CeNLuc-E2 particles produced approximately 12-fold greater
luminescence than non-immobilized CeNLuc proteins, resulting in a brighter luminescent
reporter. In contrast, LumiScarlet-E2 particles exhibited a greater red-shifted emission than
CeNLuc-E2, which could be advantageous for more sensitive deep-tissue BLI. These findings
demonstrate the potential of CeNLuc-E2 and LumiScarlet-E2 as effective reporters for broad
applications in nanomedicine and bioimaging
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IRX5 promotes DNA damage repair and activation of hair follicle stem cells
The molecular mechanisms allowing hair follicles to periodically activate their stem cells (HFSCs) are incompletely characterized. Here, we identify the transcription factor IRX5 as a promoter of HFSC activation. Irx5-/- mice have delayed anagen onset, with increased DNA damage and diminished HFSC proliferation. Open chromatin regions form near cell cycle progression and DNA damage repair genes in Irx5-/- HFSCs. DNA damage repair factor BRCA1 is an IRX5 downstream target. Inhibition of FGF kinase signaling partially rescues the anagen delay in Irx5-/- mice, suggesting that the Irx5-/- HFSC quiescent phenotype is partly due to failure to suppress Fgf18 expression. Interfollicular epidermal stem cells also show decreased proliferation and increased DNA damage in Irx5-/-mice. Consistent with a role for IRX5 as a promoter of DNA damage repair, we find that IRX genes are upregulated in many cancer types and that there is a correlation between IRX5 and BRCA1 expression in breast cancer