• Coupled systems mechanics
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• v.4 no.3
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• pp.263-277
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• 2015

This paper presents a coupled FEM-BEM strategy for the numerical analysis of elastodynamic problems where infinite-domain models and complex heterogeneous media are involved, rendering a configuration in which neither the Finite Element Method (FEM) nor the Boundary Element Method (BEM) is most appropriate for the numerical analysis. In this case, the coupling of these methodologies is recommended, allowing exploring their respective advantages. Here, frequency domain analyses are focused and an iterative FEM-BEM coupling technique is considered. In this iterative coupling, each sub-domain of the model is solved separately, and the variables at the common interfaces are iteratively updated, until convergence is achieved. A relaxation parameter is introduced into the coupling algorithm and an expression for its optimal value is deduced. The iterative FEM-BEM coupling technique allows independent discretizations to be efficiently employed for both finite and boundary element methods, without any requirement of matching nodes at the common interfaces. In addition, it leads to smaller and better-conditioned systems of equations (different solvers, suitable for each sub-domain, may be employed), which do not need to be treated (inverted, triangularized etc.) at each iterative step, providing an accurate and efficient methodology.

• BMB Reports
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• v.55 no.7
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• pp.336-341
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• 2022

Narrowing of arteries supplying blood to the limbs provokes critical hindlimb ischemia (CLI). Although CLI results in irreversible sequelae, such as amputation, few therapeutic options induce the formation of new functional blood vessels. Based on the proangiogenic potentials of stem cells, in this study, it was examined whether a combination of dental pulp stem cells (DPSCs) and human umbilical vein endothelial cells (HUVECs) could result in enhanced therapeutic effects of stem cells for CLI compared with those of DPSCs or HUVECs alone. The DPSCs+ HUVECs combination therapy resulted in significantly higher blood flow and lower ischemia damage than DPSCs or HUVECs alone. The improved therapeutic effects in the DPSCs+ HUVECs group were accompanied by a significantly higher number of microvessels in the ischemic tissue than in the other groups. In vitro proliferation and tube formation assay showed that VEGF in the conditioned media of DPSCs induced proliferation and vessel-like tube formation of HUVECs. Altogether, our results demonstrated that the combination of DPSCs and HUVECs had significantly better therapeutic effects on CLI via VEGF-mediated crosstalk. This combinational strategy could be used to develop novel clinical protocols for CLI proangiogenic regenerative treatments.

• The Korean Journal of Physiology and Pharmacology
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• v.26 no.2
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• pp.113-123
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• 2022

Diarylpropionitrile (DPN), a selective agonist for estrogen receptor β (ERβ), has been reported to regulate various hormonal responses through activation of ERβ in tissues including the mammary gland and brain. However, the effect of DPN on melanogenesis independent of ERβ has not been studied. The aim of this study is to examine the possibility of anti-melanogenic effect of DPN and its underlying mechanism. Melanin contents and cellular tyrosinase activity assay indicated that DPN inhibited melanin biosynthesis in alpha-melanocyte stimulating hormone-stimulated B16F10 melanoma cell line. However, DPN had no direct influence on in vitro tyrosinase catalytic activity. On the other hand, 17β-estradiol had no effect on inhibition of melanogenesis, suggesting that the DPN-mediated suppression of melanin production was not related with estrogen signaling pathway. Immunoblotting analysis showed that DPN down-regulated the expression of microphthalmia-associated transcription factor (MITF), a central transcription factor of melanogenesis and its down-stream genes including tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2. Also, DPN attenuated the phosphorylation of protein kinase A (PKA) and cAMP-response element-binding protein (CREB). Additionally, DPN suppressed the melanin synthesis in UVB-irradiated HaCaT conditioned media culture system suggesting that DPN has potential as an anti-melanogenic activity in physiological conditions. Collectively, our data show that DPN inhibits melanogenesis via downregulation of PKA/CREB/MITF signaling pathway.

• Oncology Letters
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• v.18 no.5
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• pp.4645-4650
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• 2019

Tumor microenvironment serves an important role in tumor growth and metastasis. Cancer cells can promote growth and malignancy by altering the surrounding stroma. Cancer-associated fibroblast (CAF) are an abundant cell type present within the tumor microenvironment and provide tumorigenic features by secreting cytokines. In the current study, the CAF-mediated invasion of oral squamous cell carcinoma (OSCC) was investigated and the associated mechanisms were elucidated. Cancer invasion was estimated using a Matrigel-coated Transwell chamber and FITC-gelatin matrix. To verify the effect of the tumor microenvironment, conditioned media (CM) from normal fibroblast (NF) and CAFs were prepared. An ELISA was performed to estimate the level of IL-1β. A proteome profiler human protease array was performed to verify the proteases affected by stimulation with CM, from CAF. Recombinant IL-1β protein increased the invasion of OSCC cells. IL-1β expression was higher in CAF than NF. CM from CAF (CM-CAF) increased cancer invasion and FITC-gelatin matrix degradation. The invasive capacity provided by CAF was abrogated by an IL-1 receptor (IL-1R) antagonist. Additionally, CM-CAF increased the secretion of ADAM 9 and Kallikrein 11 from OSCC cells. The invasion activity by CM-CAF was partially abrogated by the neutralization of ADAM 9 or Kallikrein 11. In conclusion, by providing stromal factor, CAFs were a critical inducer of OSCC invasion, and CAF secretes the required amount of IL-1β to increase cancer invasion activity. The invasive capacity of CAF was identified to be IL-1R-dependent. ADAM 9 and Kallikrein 11 were influencing factors involved in the increase of CAF-mediated cancer invasion.

• YAKHAK HOEJI
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• v.47 no.6
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• pp.410-416
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• 2003

Autotaxin(ATX) was originally purified from conditioned media of A2058 human melanoma cells and shown to be a potent cell motility-stimulating factor, possessing a type II nucleotide pyrophosphatase/phosphodiesterase (NPP2) activity. Recombinant ATX has recently demonstrated that human plasma lysophosholipase D is identical to ATX and uses lysophosphatidylcholine as a substrate to mediate various biological functions including tumor cell growth and motility through G-protein coupled receptor. However, despite pivotal roles of ATX on physiological or pathophysiological states, the production of ATX is solely depends on complicated purification method which employs multiple column steps, but resulted in very poor yield. This limited the use of ATX for extensive analysis. We, therefore, expressed six histidine-tagged recombinant human ATX(His-ATX) in High Five TM insect cells to improve the generation of ATX and to make simple the purification of ATX. The signal sequence of the human ATX gene was truncated and replaced with sequence of insect cell secretion signal within expression vector. In addition, codons for six histidines were added to the C-termini of 120kDa ATX cDNA construct. A simple purification scheme utilizing two-step affinity column chromatography was designed to purify His-ATX to homogeneity from the culture supernatant of transfected insect cells. Homogenous His-ATX was detected and isolated from the concentrated insect cell medium using concanavalin A agarose and nickel affinity chromatography. Purified His-ATX was in full length with ATX capacity. A combination of this expression system and purification scheme would be useful for production and purification of high-quality functional ATX for research and practical application of multiple functional motogen, ATX/NPP-2.

• Journal of Veterinary Science
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• v.19 no.6
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• pp.735-743
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• 2018

We investigated the effect of transforming growth factor beta 1 ($TGF-{\beta}1$) on equine hyaluronan synthase 2 (HAS2) gene expression and hyaluronan (HA) synthesis in culture models of articular chondrocytes. Equine chondrocytes were treated with $TGF-{\beta}1$ at different concentrations and times in monolayer cultures. In three-dimensional cultures, chondrocyte-seeded gelatin scaffolds were cultured in chondrogenic media containing 10 ng/mL of $TGF-{\beta}1$. The amounts of HA in conditioned media and in scaffolds were determined by enzyme-linked immunosorbent assays. HAS2 mRNA expression was analyzed by semi-quantitative reverse transcription polymerase chain reaction. The uronic acid content and DNA content of the scaffolds were measured by using colorimetric and Hoechst 33258 assays, respectively. Cell proliferation was evaluated by using the alamarBlue assay. Scanning electron microscopy (SEM), histology, and immunohistochemistry were used for microscopic analysis of the samples. The upregulation of HAS2 mRNA levels by $TGF-{\beta}1$ stimulation was dose and time dependent. $TGF-{\beta}1$ was shown to enhance HA and uronic acid content in the scaffolds. Cell proliferation and DNA content were significantly lower in $TGF-{\beta}1$ treatments. SEM and histological results revealed the formation of a cartilaginous-like extracellular matrix in the $TGF-{\beta}1$-treated scaffolds. Together, our results suggest that $TGF-{\beta}1$ has a stimulatory effect on equine chondrocytes, enhancing HA synthesis and promoting cartilage matrix generation.

• Biomolecules & Therapeutics
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• v.29 no.2
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• pp.144-153
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• 2021

Astrocytes play various important roles such as maintaining brain homeostasis, supporting neurons, and secreting inflammatory mediators to protect the brain cells. In aged subjects, astrocytes show diversely changed phenotypes and dysfunctions. But, the study of aged astrocytes or astrocytes from aged subjects is not yet sufficient to provide a comprehensive understanding of their important processes in the regulation of brain function. In this study, we induced an in vitro aged astrocyte model through late passage cultivation of rat primary cultured astrocytes. Astrocytes were cultured until passage 7 (P7) as late passage astrocytes and compared with passage 1 (P1) astrocytes as early passage astrocytes to confirm the differences in phenotypes and the effects of serial passage. In this study, we confirmed the morphological, molecular, and functional changes of late passage astrocytes showing aging phenotypes through SA-β-gal staining and measurement of nuclear size. We also observed a reduced expression of inflammatory mediators including IL-1β, IL-6, TNFα, iNOS, and COX2, as well as dysregulation of wound-healing, phagocytosis, and mitochondrial functions such as mitochondrial membrane potential and mitochondrial oxygen consumption rate. Culture-conditioned media obtained from P1 astrocytes promoted neurite outgrowth in immature primary cultures of rat cortices, which is significantly reduced when we treated the immature neurons with the culture media obtained from P7 astrocytes. These results suggest that late passage astrocytes show senescent astrocyte phenotypes with functional defects, which makes it a suitable model for the study of the role of astrocyte senescence on the modulation of normal and pathological brain aging.

• Tuberculosis and Respiratory Diseases
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• v.51 no.4
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• pp.315-324
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• 2001

Background : IL-8 is a potent chemotactic cytokine that plays an important role in the host defense mechanism against M. tuberculosis by recruiting inflammatory cells to the site of the infection. Lung epithelial cells, as well as alveolar macrophages are known to produce IL-8 in response to M. tuberculosis. IL-8 gene expression is mainly regulated on the level of transcription by NF-${\kappa}B$. This study investigated whether or not A549 cells produce IL-8 in NF-${\kappa}B$ dependent mechanism in response to macrophages phagocytosing M. tuberculosis. Methods : Peripheral blood monocytes that were obtained from healthy donors were cultured for 24 h with M. tuberculosis and a conditioned medium(CoMTB) was obtained. As a negative control, the conditioned medium without M. tuberculosis (CoMCont) was used. A549 cells were stimulated with M. tuberculosis, CoMCont and CoMTB and the IL-8 concentration in the culture media was measured by ELISA. The CoMTB induced IL-8 mRNA expression in the A549 cells was evaluated using RT-PCR, and CoMTB induced $I{\kappa}B{\alpha}$ degradation was measured using western blot analysis. CoMTB induced nuclear translocation and DNA binding of NF-${\kappa}B$ was also examined using an electrophoretic mobility shift assay(EMSA), and the CoMTB induced NF-${\kappa}B$ dependent IL-8 transcriptional activity was measured using a luciferase reporter gene assay. Results : CoMTB induced IL-8 production by A549 cells($46.8{\pm}4.8\;ng/ml$) was higher than with direct stimulation with M. tuberculosis ($6.8{\pm}2.9\;ng/ml$). CoMTB induced IL-8 mRNA expression increased after 2 h of stimulation and was sustained for 24 h. $I{\kappa}B{\alpha}$ was degraded after 10 min of CoMTB stimulation and reappeared by 60 min. CoMTB stimulated the nuclear translocation and DNA binding of NF-${\kappa}B$. The CoMTB induced NF-${\kappa}B$ dependent IL-8 transcriptional activity($13.6{\pm}4.3$ times control) was higher than either CoMCont($2.0{\pm}0.6$ times control) or M. tuberculosis ($1.4{\pm}0.6$ times control). Conclusion : A conditioned medium of peripheral blood monocytes phagocytosing M. tuberculosis stimulates NF-${\kappa}B$ dependent IL-8 production by the lung epithelial cells.

• The Journal of Korean Academy of Prosthodontics
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• v.42 no.2
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• pp.175-191
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• 2004

Statement of problem: Platelet-rich plasma(PRP) is well known to be very effective method to stimulate and accelerate the healing of bone and soft tissue. However, there are few reports which deal with the mechanisms of the PRP on the activation of the osteoblasts. Purpose: This study was aimed to investigate the effect of growth factors in PRP on the activity of osteoblasts. Material and method: To evaluate the effect on human, human osteoblast cell line was cultured. PRP was extracted from the blood of a healthy volunteer. Using the recombinant growth factors of PDGF, $TGFT-\beta$, IGF-1, bFGF which are mainly found at bone matrix and their neutralizing antibody, the effect of PRP on the attachment and proliferation of osteoblasts was evaluated. To evaluate the autocrine and paracrine effects, conditioned media(CM) of PRP was made and compared with PRP. By the western blot analysis, the expression of growth factors in PRP, CM was examined. Cell morphology was compared by the light microscope. Results : 1) The effects of CM on osteoblast were similar to the effects of PRP. 2) PRP, CM, recombinant $TGF-\beta$, bFGF, IGF-1 showed significantly higher cellular attachment than control(p<0.05) in the cell attachment assay. In the cell proliferation assay, PRP, CM, recombinant $TGF-\beta$, IGF-1, bFGF, PDGF increased significantly cell proliferation(p<0.01). Among the recombinant growth factors, IGF-1 showed the highest cellular attachment and proliferation. 3) In the western blot assay, bFGF, IGF-1, PDGF weve equally expressed in PRP and CM. 4) The attachment of osteoblast cell decreased significantly after the addition of neutralizing antibody against $TGF-\beta$, IGF-1(p<0.05). In the cell proliferation assay, the addition of neutralizing antibody against $TGF-\beta$, bFGF, PDGF, IGF-1 decreased significantly the cellular proliferation(p<0.05). The amount of decreasing in the cell attachment and proliferation is the highest in at-lGF-1. 5) The cells in control group were flattened and elongated with a few cellular processes in the a light microscope. But, the cells appeared as spherical, plump cells with well developed cellular processes in experimental groups. The cells in PRP and CM had more prominent developed features than recombinant growth factor groups. Conclusions : These findings imply that PRP maximize the cellular activity in early healing period using the synergistic effect, autocrine, paracrine effects of growth factors and increase the rate and degree of bone formation.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.10
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• pp.1403-1411
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• 2002

The aims of this study were, firstly, to analyze the biochemical compositions of serum and follicular fluid (FF) from prepubertal gilts after PMSG (1,000 IU) treatment. The concentrations of total proteins, lipids, cholesterol, glucose and sex hormones (progesterone, $P_4$; estradiol-$17{\beta}$, $E_2$; testosterone, T) were measured. Secondary, the effects of porcine FF (pFF) addition (40% and 100%) in IVM media and different culture conditions [Exp. 1: mBMOC-2+20% porcine serum (PS), fresh IVM medium, filtered IVMconditioned medium, or rabbit oviducts; Exp. 2: mBMOC-2+20%PS or stepwise medium replacement procedures (SMRP) cocultured with or without cumulus cells] on the in vitro development (IVD) of porcine oocytes were also examined. Results showed that no significant differences were found in total protein levels between serum and pFF from different sizes (large, >7 mm; medium, ~5-7 mm; small, <3-5 mm) of follicles (75-85 and 49-90 mg/dl; p>0.05). Total lipid concentrations remained constant in serum (395-472 mg/dl), and reduced significantly in the pFF from large follicles (287 mg/dl) at 132 h after PMSG treatment when compared to those at other time points (441-480 mg/dl). Basal cholesterol levels in serum and pFF at 12 h were similar (153-161 mg/dl), but increased at 36 h (186-197 mg/dl). Basal P4 and E2 levels in serum (0.1 ng/ml and 5.5 pg/ml) were low, but increased from 0.34 ng/ml and 12.13 pg/ml at 24 h to 0.81 ng/ml and 61.70 pg/ml at 98 h, respectively, after PMSG treatment (p<0.05). P4 levels increased linearly in pFF from large follicles during 12 through 132 h (138-1,288 ng/ml). A similar increase was also observed in $E_2$ levels (22-730 pg/ml) before 60 h post PMSG treatment, and then dropped afterwards (730-121 pg/ml). The development of the oocytes fertilized in 40% pFF-medium was greater than that in 100% pFF-medium group without gonaodtropin addition (31% vs 10%, p<0.05). However, both were lower than those in mBMOC-2+20%PS and in rabbit oviducts (p<0.05). When cocultured with cumulus cell monolayers, a greater cleavage rate was observed in the group cultured in filtered IVM-conditioned medium than the SMRP group (36% vs 18%, p<0.05). A similar phenomenon was also observed in the culture without cumulus cell monolayers (33% vs 19%, p<0.05). It is concluded that neither the fresh IVM nor filtered IVM-conditioned medium has positive effect on the IVD of oocytes. Coculture with cumulus cell monolayers and the SMRP were not beneficial to the development of IVF pig oocytes.