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Quantum Dot®s (QDot®s) are novel, semi-conductive nanostructures that emit a certain fluorescence when excited by specific wavelengths. QDot®s are more photostable, brighter, and photobleach less than other fluorescent dyes. These characteristics give them the potential to be used in many biological applications. The shells of QDot®s are coated with functional groups, such as carboxylate and organic groups, allowing them to couple to peptides/proteins and be used for real-time imaging and high-resolution microscopy. Here, we utilize Quantum Dot®s and Bone Morphogenetic Protein-2 (BMP-2) to create a BMP-2-QDot®s conjugate. BMP-2 is a growth factor that drives many processes such as cardiogenesis, neural growth, and osteogenesis. Despite its numerous roles, the trafficking and uptake of BMP-2 into cells is not well-established, especially during progression of diseases. The results presented here demonstrate for the first time a fluorescent BMP-2 analog that binds to the BMP-receptors (BMPRs), remains biologically active, and is stable for long time periods. Previous attempts to develop a biological BMP-2 analog with Fluorescein isothiocyanate (FITC) or nanodiamonds lacked data on the analog’s stability. Furthermore, these analogs did not address whether they can signal within the cell by binding to the BMPRs or were mediated by non-stable conjugates.

Background: Osteoporosis is a degenerative skeletal disease with a limited number of treatment options. CK2.3, a novel peptide, may be a potential therapeutic. It induces osteogenesis and bone formation in vitro and in vivo by acting downstream of BMPRIA through releasing CK2 from the receptor. However, the detailed signaling pathways, the time frame of signaling, and genes activated remain largely unknown. Methods: Using a newly developed fluorescent CK2.3 analog, specific inhibitors for the BMP signaling pathways, Western blot, and RT-qPCR, we determined the mechanism of CK2.3 in C2C12 cells. We then confirmed the results in primary BMSCs. Results: Using these methods, we showed that CK2.3 stimulation activated OSX, ALP, and OCN. CK2.3 stimulation induced time dependent release of CK2β from BMPRIA and concurrently CK2.3 colocalized with CK2α. Furthermore, CK2.3 induced BMP signaling depends on ERK1/2 and Smad1/5/8 signaling pathways. Conclusion: CK2.3 is a novel peptide that drives osteogenesis, and we detailed the molecular sequence of events that are triggered from the stimulation of CK2.3 until the induction of mineralization. This knowledge can be applied in the development of future therapeutics for osteoporosis.

Osteoporosis is a debilitating skeletal disorder that is characterized by loss of bone densityover time. It affects one in two women and one in four men, age 50 and older. New treatmentsthat specifically drive bone formation are desperately needed. We developed a peptide, CK2.3, thatacts downstream of the bone morphogenetic protein receptor type Ia and it induces osteogenesisin-vitro and in-vivo. However, its mechanism of action, especially its mode of uptake by cellsremains unknown. To demonstrate CK2.3 internalization within a cell, we conjugated CK2.3to Quantum Dot®s (Qdot®s), semiconductor nanoparticles. We purified CK2.3-Qdot®s by sizeexclusion chromatography and verified the conjugation and stability using UV/VIS and Fouriertransform infrared spectroscopy. Our results show that CK2.3 was conjugated to the Qdot®s andthe conjugate was stable for at least 4 days at 37 °C. Moreover, CK2.3-Qdot®s exerted biologicalresponse similar to CK2.3. Addition of CK2.3-Qdot®s to cells followed by confocal imaging revealedthat CK2.3-Qdot®s were internalized at 6 h post stimulation. Furthermore, using pharmacologicalinhibitors against endocytic pathways, we demonstrated that CK2.3-Qdot®s were internalized bycaveolae. These results show for the first time that the novel peptide CK2.3 is taken up by the cellthrough caveolae mediated endocytosis.

Osteoporosis is a debilitating skeletal disorder that is characterized by loss of bone density over time. It affects one in two women and one in four men, age 50 and older. New treatments that specifically drive bone formation are desperately needed. We developed a peptide, CK2.3, that acts downstream of the bone morphogenetic protein receptor type Ia and it induces osteogenesis in-vitro and in-vivo. However, its mechanism of action, especially its mode of uptake by cells remains unknown. To demonstrate CK2.3 internalization within a cell, we conjugated CK2.3 to Quantum Dot®s (Qdot®s), semiconductor nanoparticles. We purified CK2.3-Qdot®s by size exclusion chromatography and verified the conjugation and stability using UV/VIS and Fourier transform infrared spectroscopy. Our results show that CK2.3 was conjugated to the Qdot®s and the conjugate was stable for at least 4 days at 37 °C. Moreover, CK2.3-Qdot®s exerted biological response similar to CK2.3. Addition of CK2.3-Qdot®s to cells followed by confocal imaging revealed that CK2.3-Qdot®s were internalized at 6 h post stimulation. Furthermore, using pharmacological inhibitors against endocytic pathways, we demonstrated that CK2.3-Qdot®s were internalized by caveolae. These results show for the first time that the novel peptide CK2.3 is taken up by the cell through caveolae mediated endocytosis. [ABSTRACT FROM AUTHOR]

Piezo channels are associated with neuropathology in diseases like traumatic brain injury and glaucoma, but pathways linking tissue stretch to aberrant neural signaling remain unclear. The present study demonstrates that Piezo1 activation increases action potential frequency in response to light and the spontaneous dark signal from mouse retinal explants. Piezo1 stimulation was sufficient to increase cytoplasmic Ca 2+ in soma and neurites, while stretch increased spiking activity in current clamp recordings from of isolated retinal ganglion cells (RGCs). Axon-marker beta-tubulin III colocalized with both Piezo1 and Piezo2 protein in the mouse optic nerve head, while RGC nuclear marker BRN3A colocalized with Piezo channels in the soma. Piezo1 was also present on GFAP-positive regions in the optic nerve head and colocalized with glutamine synthetase in the nerve fiber layer, suggesting expression in optic nerve head astrocytes and Müller glia end feet, respectively. Human RGCs from induced pluripotent stem cells also expressed Piezo1 and Piezo2 in soma and axons, while staining patterns in rats resembled those in mice. mRNA message for Piezo1 was greatest in the RPE/choroid tissue, while Piezo2 levels were highest in the optic nerve, with both channels also expressed in the retina. Increased expression of Piezo1 and Piezo2 occurred both 1 and 10 days after a single stretch in vivo; this increase suggests a potential role in rising sensitivity to repeated nerve stretch. In summary, Piezo1 and Piezo2 were detected in the soma and axons of RGCs, and stimulation affected the light-dependent output of RGCs. The rise in RGCs excitability induced by Piezo stimulation may have parallels to the early disease progression in models of glaucoma and other retinal degenerations., Highlights: Activation of Piezo1 excites retinal ganglion cells, paralleling the early neurodegenerative progression in glaucoma mouse models and retinal degeneration.Piezo1 and Piezo2 were expressed in axons and soma of retinal ganglion cells in mice, rats, and human iPSC-RGCs.Functional assays confirmed Piezo1 in soma and neurites of neurons. Sustained elevation of Piezo1 and Piezo2 occurred after a single transient stretch may enhance damage from repeated traumatic nerve injury.

Quantum Dot ® s (QDot ® s) are novel, semi-conductive nanostructures that emit a certain fluorescence when excited by specific wavelengths. QDot ® s are more photostable, brighter, and photobleach less than other fluorescent dyes. These characteristics give them the potential to be used in many biological applications. The shells of QDot ® s are coated with functional groups, such as carboxylate and organic groups, allowing them to couple to peptides/proteins and be used for real-time imaging and high-resolution microscopy. Here, we utilize Quantum Dot ® s and Bone Morphogenetic Protein-2 (BMP-2) to create a BMP-2-QDot ® s conjugate. BMP-2 is a growth factor that drives many processes such as cardiogenesis, neural growth, and osteogenesis. Despite its numerous roles, the trafficking and uptake of BMP-2 into cells is not well-established, especially during progression of diseases. The results presented here demonstrate for the first time a fluorescent BMP-2 analog that binds to the BMP-receptors (BMPRs), remains biologically active, and is stable for long time periods. Previous attempts to develop a biological BMP-2 analog with Fluorescein isothiocyanate (FITC) or nanodiamonds lacked data on the analog's stability. Furthermore, these analogs did not address whether they can signal within the cell by binding to the BMPRs or were mediated by non-stable conjugates.