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(searched for: doi:10.1126/stke.4172007pl7)
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Published: 23 November 2019
by MDPI
International Journal of Molecular Sciences, Volume 20; https://doi.org/10.3390/ijms20235877

Abstract:
Bone is one of the most important organs in the human body. It provides structure, function, and protection for other vital organs; therefore, bone maintenance and homeostasis are critical processes. As humans age, their bone mineral density decreases, which leads to diseases like osteoporosis. This disease affects one in two women and one in five men aged 50 and over. As the aging population increases, the interest and significance of studying this debilitating bone disease becomes more relevant. Current therapeutic products for osteoporosis have many side effects and can be taken for a limited number of years. Most therapeutic products only focus on decreasing bone resorption, not increasing bone formation. Bone morphogenetic protein 2 is an essential growth factor that drives osteoblast differentiation and activity and is essential for bone formation. However, usage in the clinic is unsuccessful due to several side effects. Recently, a signaling disparity in bone marrow stromal cells within the bone morphogenetic protein pathway that led to decreased bone morphogenetic protein 2 responsiveness was identified in patients diagnosed with osteoporosis. However, it is unclear how other cell populations, especially osteoblasts, which are key players in bone remodeling, are affected and whether the bone morphogenetic protein pathway is affected during osteoporosis. Our research group designed a novel peptide, casein kinase 2.3, that acts downstream of the bone morphogenetic receptor type Ia and increases bone mineralization in murine cells and primary bovine osteoblasts. The aim of the study presented here was to compare the responsiveness of osteoblasts to bone morphogenetic protein 2 and casein kinase 2.3, especially in patients diagnosed with osteoporosis. Mature osteoblasts were extracted from patients diagnosed with osteoporosis or osteoarthritis from Christiana Care Hospital in Newark, Delaware. They were stimulated with either bone morphogenetic protein 2 or casein kinase 2.3, and their effect on osteoblast activity was determined. The osteoporotic patients showed no mineralization response to bone morphogenetic protein 2 stimulation, while the osteoarthritis patients significantly responded to bone morphogenetic protein 2 stimulation. Furthermore, markers for osteoblast activity were increased by casein kinase 2.3, which was in sharp contrast to bone morphogenetic protein 2. This further supports a major bone morphogenetic protein signaling disparity in both the elderly and those suffering with osteoporosis. Both patient types did significantly respond to casein kinase 2.3. Further analysis of the bone morphogenetic protein pathway could lead to new therapeutic products for osteoporosis.
Published: 9 July 2018
by MDPI
Nanomaterials, Volume 8; https://doi.org/10.3390/nano8070513

Abstract:
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.
John Nguyen, Hilary Weidner, Lora M. Schell, Linda Sequeira, Ryan Kabrick, Saurabh Dharmadhikari, Harold Coombs, Randall L. Duncan, Liyun Wang,
Published: 1 January 2018
Journal of Bone Research, Volume 6; https://doi.org/10.4172/2572-4916.1000190

Abstract:
Osteoporosis is a silent disease caused by low bone mineral density that results in bone fractures in 1 out of 2 women and 1 in 4 men over the age of 50. Although several treatments for osteopenia and osteoporosis are available, they have severe side effects and new treatments are desperately needed. Current treatments usually target osteoclasts and inhibit their activity or differentiation. Treatments that decrease osteoclast differentiation and activity but enhance osteogenesis and osteoblast activity are not available. We recently developed a peptide, CK2.3, that induces bone formation and increases bone mineral density as demonstrated by injection over the calvaria of 6 to 9-day-old mice and tail vein injection of 8-week-old mice. CK2.3 also decreased osteoclast formation and activity. However, these studies raise questions: does CK2.3 induce similar results in old mice and if so, what is the effective CK2.3 concentration and, is the bone mineral density of vertebrae of the spinal column increased as well? CK2.3 was systematically injected into the tail vein of female 6-month old mice with various concentrations of CK2.3: 0.76 μg/kg, 2.3 μg/kg, or 6.9 μg/kg per mice. Mice were sacrificed one week, two weeks, and four weeks after the first injection. Their spines and femurs were collected and analyzed for bone formation. Femur and lumbar spine analyses found increased bone mineral density (BMD) and mineral apposition rate, with greater stiffness observed in femoral samples four weeks after the first injection. Histochemistry showed that osteoclastogenesis was suppressed in CK2.3 treated senile mice. For the first time, this study showed the increase of lumbar spine BMD by CK2.3. Moreover, it showed that enhancement of femur BMD was accompanied by increased femur stiffness only at medium concentration of CK2.3 four weeks after the first injection indicating the maintenance of bone’s structural integrity by CK2.3.
Hemanth Akkiraju, Padma Pradeepa Srinivasan, Xian Xu, Xinqiao Jia, Catherine B. Kirn Safran,
Published: 18 April 2017
Stem Cell Research & Therapy, Volume 8, pp 1-11; https://doi.org/10.1186/s13287-017-0537-y

Abstract:
Osteoarthritis (OA) of the knee involves degeneration of articular cartilage of the diarthrodial joints. Current treatment options temporarily relieve the joint pain but do not restore the lost cartilage. We recently designed a novel bone morphogenetic protein receptor type I (BMPRI) mimetic peptide, CK2.1, that activates BMPRIa signaling in the absence of bone morphogenetic protein (BMP). Our previous research demonstrated that CK2.1 induced chondrogenesis in vitro and in vivo; however, it is unknown if CK2.1 restores damaged articular cartilage in vivo. In this study, we demonstrate that CK2.1 induced articular cartilage (AC) repair in an OA mouse model. We designed hyaluronic acid (HA)-based hydrogel particles (HGPs) that slowly release CK2.1. HGP-CK2.1 particles were tested for chondrogenic potency on pluripotent mesenchymal stem cells (C3H10T1/2 cells) and locally injected into the intra-articular capsule in mice with cartilage defects. C57BL/6J mice were operated on to destabilize the medial meniscus and these mice were kept for 6 weeks after surgery to sustain OA-like damage. Mice were then injected via the intra-articular capsule with HGP-CK2.1; 4 weeks after injection the mice were sacrificed and their femurs were analyzed for cartilage defects. Immunohistochemical analysis of the cartilage demonstrated complete repair of the AC compared to sham-operated mice. Immunofluorescence analysis revealed collagen type IX production along with collagen type II in the AC of mice injected with HGP-CK2.1. Mice injected with phosphate-buffered saline (PBS) and HGP alone had greater collagen type X and osteocalcin production, in sharp contrast to those injected with HGP-CK2.1, indicating increased chondrocyte hypertrophy. Our results demonstrate that the slow release HGP-CK2.1 drives cartilage repair without the induction of chondrocyte hypertrophy. The peptide CK2.1 could be a powerful tool in understanding the signaling pathways contributing to the repair process, and also may be used as a potential therapeutic for treating degenerative cartilage diseases such as OA.
Hemanth Akkiraju, Jeremy Bonor,
Journal of Orthopaedic Research, Volume 35, pp 876-885; https://doi.org/10.1002/jor.23342

Abstract:
Bone morphogenetic protein 2 regulates chondrogenesis and cartilage formation. However, it also induces chondrocyte hypertrophy and cartilage matrix degradation. We recently designed three peptides CK2.1, CK2.2, and CK2.3 that activate the BMP signaling pathways by releasing casein kinase II (CK2) from distinct sites at the bone morphogenetic protein receptor type Ia (BMPRIa). Since BMP2 is a major regulator of chondrogenesis and the peptides activated BMP signaling in a similar way, we evaluated the effect of these peptides on chondrogenesis and cartilage formation. C3H10T1/2 cells were stimulated with CK2.1, CK2.2, and CK2.3 and evaluated for the chondrogenic and osteogenic potential. For chondrogenesis, Alcian blue staining was performed. Additionally, collagen types II and X expression was measured. For osteogenesis, osteocalcin and von Kossa staining were performed. From the three peptides, CK2.1 was the most promising peptide to induce chondrogenesis but not osteogenesis. To investigate the effect of CK2.1 on articular cartilage formation in vivo, we injected CK2.1 into the tail vein of mice. Injection of CK2.1 into the tail vein of mice led to increased articular cartilage formation but not BMD. In sharp contrast, injection of BMP2 led to increased BMD and expression of collagen type X, a marker of chondrocyte hypertrophy. MMP13 expression was unchanged. Our study demonstrates that CK2.1 drives chondrogenesis and cartilage formation without induction of chondrocyte hypertrophy. Peptide CK2.1 may, therefore, be a valuable therapeutic for cartilage degenerative diseases. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:876–885, 2017.
Titiwat Sungkaworn, Chutima Jiarpinitnun, Pongkorn Chaiyakunvat, Varanuj Chatsudthipong
European Journal of Medicinal Chemistry, Volume 63, pp 629-634; https://doi.org/10.1016/j.ejmech.2013.02.041

The publisher has not yet granted permission to display this abstract.
Oleksandra Moseychuk, Hemanth Akkiraju, Joyita Dutta, Alex D’Angelo, , Randall L. Duncan,
Journal of Cell Communication and Signaling, Volume 7, pp 265-278; https://doi.org/10.1007/s12079-013-0199-1

The publisher has not yet granted permission to display this abstract.
Published: 1 August 2012
by SPIE
Journal of Biomedical Optics, Volume 17, pp 0808011-0808017; https://doi.org/10.1117/1.jbo.17.8.080801

Abstract:
Abstract. Image correlation spectroscopy (ICS) is a powerful technique for detecting arrangement of fluorophores in images. This tutorial gives background into the mathematical underpinnings of ICS, specifically image autocorrelation. The effects of various artifacts and image processing steps, including background subtraction, noise, and image morphology were examined analytically and their effects on ICS analysis modeled. A series of recommendations was built based on this analysis.
, Alex D'Angelo, Lauren Gurski, Jeremy Bonor, Kathryn L. Schultz, Wesley G. Beamer, Clifford J. Rosen,
Published: 31 January 2012
Bone, Volume 50, pp 189-199; https://doi.org/10.1016/j.bone.2011.10.016

The publisher has not yet granted permission to display this abstract.
Sadiq Mohammed Mir, Brenda Baggett,
Published: 3 January 2012
Biomedical Optics Express, Volume 3, pp 215-224; https://doi.org/10.1364/BOE.3.000215

Abstract:
Image correlation spectroscopy (ICS) is known to be a useful tool for the evaluation of fiber width in the extracellular matrix. Here we evaluate a more general from of ICS fit parameters for fiber networks and arrive at a means of quantifying the fiber density, pore size and length which facilitates the characterization of the extracellular matrix. A simulation package was made to create images with different structural properties of fiber networks such as fiber orientation, width, fiber density and length. A pore finding algorithm was developed which determines the distribution of circular voids in the image. Collagen I hydrogels were prepared under different polymerization conditions for validation of our pore size algorithm with microscopy data. ICS parameters included amplitude, standard deviation and ellipticity and are shown to predict the structural properties of fiber networks in a quantitative manner. While the fiber width is related to the ICS sigma; the fiber density relates to the pore size distribution which correlates with the ICS amplitude in thresholded images. Fiber length is related to ICS ellipticity if the fibers have a preferred orientation. Findings from ICS and pore distribution algorithms were verified for both simulated and microscopy data. Based on these findings, we conclude that ICS can be used in the assessment of the extracellular matrix and the prediction of fiber orientation, width, density, length and matrix pore size.
, Jeremy Bonor, Kathryn L. Shultz, Wesley G. Beamer, Clifford J. Rosen,
Published: 14 December 2011
Journal of Cellular Physiology, Volume 227, pp 2870-2879; https://doi.org/10.1002/jcp.23028

The publisher has not yet granted permission to display this abstract.
Titiwat Sungkaworn, Yongwimon Lenbury, Varanuj Chatsudthipong
Biochimica et Biophysica Acta (BBA) - Biomembranes, Volume 1808, pp 2496-2500; https://doi.org/10.1016/j.bbamem.2011.07.007

The publisher has not yet granted permission to display this abstract.
Sally A. Kim, , Michelle A. Digman, Enrico Gratton, Petra Schwille, ,
The Journal of Neuroscience, Volume 30, pp 16409-16416; https://doi.org/10.1523/JNEUROSCI.3063-10.2010

Abstract:
Translational mobility is involved in every process in neurobiology—released neurotransmitter diffuses through the synaptic cleft in search of receptor targets, membrane receptors traffic to synaptic sites in the neuron, RNA and other cargo are transported to distal dendrites, and cell signaling
, Shayamala Thinakaran, Oleksandra Moseychuk, Daniel King, Kira Young, David W. Litchfield, Nils O. Petersen,
Published: 4 August 2010
Biophysical Journal, Volume 99, pp 897-904; https://doi.org/10.1016/j.bpj.2010.04.070

The publisher has not yet granted permission to display this abstract.
, Mansoureh Eghbali, Jimmy Ou, Rong Lu, Ligia Toro, Enrico Stefani
Published: 3 February 2010
Biophysical Journal, Volume 98, pp 493-504; https://doi.org/10.1016/j.bpj.2009.10.037

Abstract:
To quantify spatial protein-protein proximity (colocalization) in paired microscopic images of two sets of proteins labeled by distinct fluorophores, we showed that the cross-correlation and the autocorrelation functions of image intensity consisted of fast and slowly decaying components. The fast component resulted from clusters of proteins specifically labeled, and the slow component resulted from image heterogeneity and a broadly-distributed background. To better evaluate spatial proximity between the two specifically labeled proteins, we extracted the fast-decaying component by fitting the sharp peak in correlation functions to a Gaussian function, which was then used to obtain protein-protein proximity index and the Pearson's correlation coefficient. We also employed the median-filter method as a universal approach for background reduction to minimize nonspecific fluorescence. We illustrated our method by analyzing computer-simulated images and biological images.
, Shayamala Thinakaran, Jeremy Bonor, T. Michael Underhill, Nils O. Petersen,
Published: 2 September 2009
Biophysical Journal, Volume 97, pp 1428-1435; https://doi.org/10.1016/j.bpj.2009.05.061

The publisher has not yet granted permission to display this abstract.
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