Microarrays Incorporating Gold Grid Patterns for Protein Quantification.

Protein microarrays are miniaturized two-dimensional arrays, incorporating thousands of immobilized proteins, typically printed in minute amounts on functionalized solid substrates, which can be analyzed in a high-throughput fashion. Irreproducibility of the printing techniques adopted, resulting in inconsistently and nonuniformly deposited microscopic spots, nonuniform signal intensities from the printed microspots, and significantly high background noise are some of the critical issues that affect protein analysis using traditional protein microarrays. To overcome such issues, in this study, we introduced a novel gold grid pattern-based protein microarray. The grid patterns incorporated in our microarray are equivalent to the spots used for protein analysis in conventional protein microarrays. We utilized the signal intensities from the grid patterns acting as spots for quantifying the protein concentration levels. To demonstrate the utility of our novel design concept, we quantified as low as 66.7 ng/mL of bovine serum albumin using our gold grid pattern-based protein microarray. Our grid pattern-based design concept for protein quantification overcame the signal nonuniformity issues and ensured that the dominance of any distorted signal from a single spot did not affect the overall protein quantification results as encountered in conventional protein microarrays.

Local administration of nuclear factor of activated T cells (NFAT) c1 inhibitor to suppress early resorption and inflammation induced by bone morphogenetic protein-2.

Nuclear factor of activated T cells (NFAT)-c1 is known as a key regulator in osteoclast differentiation and immune response. This study is a follow-up to our previous study showing the antiresorptive activity of VIVIT, a peptide type NFATc1 inhibitor, using absorbable collagen sponge (ACS). This study aimed to investigate the effective concentration range of local VIVIT that suppresses early excessive osteoclast activation and inflammation induced by high-dose recombinant human bone morphogenetic protein (rhBMP)-2 and concomitantly enhances bone healing in a rat critical-sized calvaria defect model. High-dose rhBMP-2 (40 µg/defect) alone significantly increased in vivo osteoclast activation and expression of the inflammatory cytokines interleukin-1ß and transforming necrosis factor-α on the scaffold at 7 days after surgery. However, rhBMP-2 had no direct effect on osteoclast activation in vitro. Osteoclast activation by rhBMP-2 was significantly suppressed by combined treatment with VIVIT at concentrations of 75 and 150 µM, but not at 15 µM, whereas suppression of inflammation occurred at all doses of VIVIT. Microcomputed tomography at 4 and 8 weeks after implantation revealed that the combination of rhBMP-2 and VIVIT at 75 µM VIVIT led to a greater bone fraction at the initial defect area, compared with rhBMP-2 alone. These findings revealed that local administration of VIVIT at certain concentrations has multiple positive effects that weaken early excessive osteoimmunological responses and enhance bone healing after rhBMP-2 administration. VIVIT has the potential to expand the therapeutic area of high-dose rhBMP-2 therapy to inflammatory bone loss. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1299-1310, 2018.

Impaired motor coordination in Pitx3 overexpression mice.

Midbrain dopaminergic (DA) neurons are involved in the regulation of voluntary movement and in emotion-related behaviors and are affected in Parkinson's disease (PD). The homeodomain transcription factor Pitx3, which is uniquely expressed in midbrain DA neurons, plays a critical role in the development, function and maintenance of midbrain DA neurons. Pitx3 deficiency results in selective deficits of midbrain DA neurons in the substantia nigra pars compacta (SNc), reminiscent of the specific DA neuronal loss observed in PD. In this study, we found that selective overexpression of Pitx3 in intact midbrain DA neurons significantly affects the function of midbrain DA neurons. We observed changes in DA levels and gene expressions in mice overexpressing Pitx3. Furthermore, motor coordination and locomotion activities are significantly affected in mice overexpressing Pitx3, suggesting that the expression level of Pitx3 plays an important role in the function of midbrain DA neuron in vivo.

Egr1 mediated the neuronal differentiation induced by extremely low-frequency electromagnetic fields.

AIM: There is a specific frequency of extremely low-frequency electromagnetic field (ELF-EMF) that promotes neuronal differentiation. Although several mechanisms are known to regulate ELF-EMF-induced neuronal differentiation, a key factor that mediates neurogenic potentials by the ELF-EMF is largely unknown. Also, the potential use of ELF-EMF exposure in cell transplantation assays is yet to be determined, including their possible use in ELF-EMF based therapy of neurological diseases. The aim of this study is to understand the underlying mechanisms that mediate ELF-EMF-induced neuronal differentiation and also to harness these mechanisms for cell transplantation assays. MAIN METHOD: Human bone marrow-mesenchymal stem cells (hBM-MSCs) were exposed to ELF-EMF (50 Hz frequency, 1mT intensity) for 8 days. The hBM-MSC derived neurons were then analyzed by general molecular biology techniques including immunofluorescence and quantitative RT-PCR. To assess changes in gene expression induced by ELF-EMF exposure, we analyzed the transcriptome of neuronal cells after an 8-day ELF-EMF exposure (50 Hz, 1 mT) and compared the transcriptional profiles to control cells. KEY FINDING: We found that early growth response protein 1 (Egr1) is one of the key transcription factors in ELF-EMF-induced neuronal differentiation. In addition, we show that transplantations of ELF-EMF-induced neurons significantly alleviate symptoms in mouse models of neurodegenerative disease. SIGNIFICANCE: These findings indicate that a specific transcriptional factor, Egr1, mediates ELF-EMF-induced neuronal differentiations, and demonstrate the promise of ELF-EMF based cell replacement therapies for neurodegenerative diseases.