• Tissue Engineering and Regenerative Medicine
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• v.15 no.6
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• pp.721-733
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• 2018

BACKGROUND: Because three-dimensional (3D) models more closely mimic native tissues, one of the goals of 3D in vitro tissue models is to aid in the development and toxicity screening of new drug therapies. In this study, a 3D skin wound healing model comprising of a collagen type I construct with fibrin-filled defects was developed. METHODS: Optical imaging was used to measure keratinocyte migration in the presence of fibroblasts over 7 days onto the fibrin-filled defects. Additionally, cell viability and growth of fibroblasts and keratinocytes was measured using the $alamarBlue^{(R)}$ assay and changes in the mechanical stiffness of the 3D construct was monitored using compressive indentation testing. RESULTS: Keratinocyte migration rate was significantly increased in the presence of fibroblasts with the cells reaching the center of the defect as early as day 3 in the co-culture constructs compared to day 7 for the control keratinocyte monoculture constructs. Additionally, constructs with the greatest rate of keratinocyte migration had reduced cell growth. When fibroblasts were cultured alone in the wound healing construct, there was a 1.3 to 3.4-fold increase in cell growth and a 1.2 to 1.4-fold increase in cell growth for keratinocyte monocultures. However, co-culture constructs exhibited no significant growth over 7 days. Finally, mechanical testing showed that fibroblasts and keratinocytes had varying effects on matrix stiffness with fibroblasts degrading the constructs while keratinocytes increased the construct's stiffness. CONCLUSION: This 3D in vitro wound healing model is a step towards developing a mimetic construct that recapitulates the complex microenvironment of healing wounds and could aid in the early studies of novel therapeutics that promote migration and proliferation of epithelial cells.

• Advances in Traditional Medicine
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• v.10 no.4
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• pp.263-270
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• 2010

Scoparia dulcis L. is widely used in the traditional system of medicine for treating liver ailments. In the present study the terpenoids and terpenoid fractions isolated from 1:1:1 petroleum ether, diethyl ether and methanol (PDM) extract of Scoparia dulcis L. were tested for their in vitro 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. Selected samples from the assay were further tested for their in vitro hepatoprotective activity against $CCl_4$ induced hepatotoxicity in freshly isolated rat hepatocytes. In the in vitro antioxidant study, fractions 7, 11, 13, 14, and 15 and PDM extract show the DPPH radical scavenging activity. The phytochemical screening of all these fractions show the presence of terpenoids. In the in vitro hepatoprotective study all these fractions and the PDM extract significantly prevent the $CCl_4$ induced changes in the aspartate aspartate amino transferase, alanine amino transferase and alkaline phosphatase levels (p < 0.05). The above results are comparable with the standard, silymarin. The results of the study indicate that, the PDM extract of Scoparia dulcis L. possesses potential hepatoprotective activity and this may be attributed to its free radical scavenging potential, which in turn may be attributed to the presence of terpenoids.

• Clinical and Experimental Reproductive Medicine
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• v.44 no.1
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• pp.40-46
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• 2017

Objective: To describe in vitro development of human embryos derived from an individual with a homozygous pathogenic variant in NLRP7 (19q13.42) and recurrent hydatidiform mole (HM), an autosomal recessive condition thought to occur secondary to an oocyte defect. Methods: A patient with five consecutive HM pregnancies was genomically evaluated via next generation sequencing followed by controlled ovarian hyperstimulation, in vitro fertilization (IVF) with intracytoplasmic sperm injection, embryo culture, and preimplantation genetic screening. Findings in NLRP7 were recorded and embryo culture and biopsy data were tabulated as a function of parental origin for any identified ploidy error. Results: The patient was found to have a pathogenic variant in NLRP7 (c.2810+2T>G) in a homozygous state. Fifteen oocytes were retrieved and 10 embryos were available after fertilization via intracytoplasmic sperm injection. Developmental arrest was noted for all 10 embryos after 144 hours in culture, thus no transfer was possible. These non-viable embryos were evaluated by karyomapping and all were diploid biparental; two were euploid and eight had various aneuploidies all of maternal origin. Conclusion: This is the first report of early human embryo development from a patient with any NLRP7 mutation. The pathogenic variant identified here resulted in global developmental arrest at or before blastocyst stage. Standard IVF should therefore be discouraged for such patients, who instead need to consider oocyte (or embryo) donation with IVF as preferred clinical methods to treat infertility.

• Parasites, Hosts and Diseases
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• v.53 no.4
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• pp.385-394
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• 2015

Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain. Next, we evaluated the sensitivity of these parasites to amphotericin B (AmB) as a standard drug in 2 parasitic phases, promastigote and amastigote. This comparison was made by MTT and nitric oxide (NO) assay and by quantifying the specific signals derived from reporter genes like EGFP intensity and luciferase activity. To study the amastigote form, both B10R and THP-1 macrophage cell lines were infected in the stationary phase and were exposed to AmB at different time points. Our results clearly revealed that the 3 parasite lines had similar in vitro infectivity rates with comparable parasite-induced levels of NO following interferon-${\gamma}$/lipopolysaccharide induction. Based on our results we proposed the more reporter gene, the faster and more sensitive evaluation of the drug efficiency.

• Korean Journal of Veterinary Research
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• v.63 no.4
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• pp.33.1-33.5
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• 2023

Many scolicidal agents have been used to destroy fertile protoscolices, but these scolicidal agents have side effects, highlighting the need for research on effective and non-toxic replacement scolicidal agents. Gold nanoparticles (AuNPs) are biocompatible and non-toxic. The current study examined the effects of AuNPs in killing the protoscolices of Echinococcus granulosus in vitro using eosin staining. The protoscolices were treated with 0.2, 0.4, 0.8, or 1.0 mg/mL of AuNPs for 15, 30, 45, or 60 minutes. A concentration of 1.0 mg/mL was the most efficient in killing the protoscolices after 60 minutes exposure, reaching 96%, followed by 0.8 mg/mL (84.5%), whereas 0.4 and 0.2 mg/mL of AuNPs achieved a death rate of 76.8% and 68.5%, respectively. The loss of the protoscolices was lower at shorter exposure times with the same concentration of AuNPs and increased as the AuNP concentration was increased at the same exposure time. Significant differences were found between the different groups compared to the control group.

• Journal of Pharmaceutical Investigation
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• v.33 no.2
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• pp.105-112
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• 2003

Selective COX (cyclooxygenase)-2 inhibitors including celecoxib have been shown to induce apoptosis and cell cycle changes in various tumor cells. New inhibitors are recently being developed as chemomodulating agents. We evaluated celecoxib and screened 150 synthetic compounds for anti-proliferative activities in vitro. Effects of celecoxib on COX activity, cell growth, cell cycle distribution, and apoptosis induction were determined in A549 COX-2 overexpressing human non-small cell lung cancer (NSCLC) cells. The COX inhibition of celecoxib increased with concentration up to 82% at $1\;{\mu}M$ after 24 hr exposure. Forty ${\mu}M$ and $50\;{\mu}M$ of ce1ecoxib induced $G_1$ arrest, and TUNEL-positive apoptotic cells, respectively. Among 150 compounds, several compounds were selected for having greater COX-2 inhibitory activity and higher selectivity than celecoxib with growth inhibitory activity. Celecoxib showed concentration-dependent COX inhibitory activity, and ability to induce cell cycle arrest and apoptosis in human NSCLC cells in vitro. Among synthetic analogues screened, several compounds showed promising in vitro activity as COX-2 inhibitory anticancer agents, which warrant further evaluation in vitro and in vivo.

• Molecules and Cells
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• v.43 no.6
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• pp.517-529
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• 2020

Carboxyl-terminal binding proteins (CtBPs) are transcription regulators that control gene expression in multiple cellular processes. Our recent findings indicated that overexpression of CtBP2 caused the repression of multiple bone development and differentiation genes, resulting in atrophic nonunion. Therefore, disrupting the CtBP2-associated transcriptional complex with small molecules may be an effective strategy to prevent nonunion. In the present study, we developed an in vitro screening system in yeast cells to identify small molecules capable of disrupting the CtBP2-p300 interaction. Herein, we focus our studies on revealing the in vitro and in vivo effects of a small molecule NSM00158, which showed the strongest inhibition of the CtBP2-p300 interaction in vitro. Our results indicated that NSM00158 could specifically disrupt CtBP2 function and cause the disassociation of the CtBP2-p300-Runx2 complex. The impairment of this complex led to failed binding of Runx2 to its downstream targets, causing their upregulation. Using a mouse fracture model, we evaluated the in vivo effect of NSM00158 on preventing nonunion. Consistent with the in vitro results, the NSM00158 treatment resulted in the upregulation of Runx2 downstream targets. Importantly, we found that the administration of NSM00158 could prevent the occurrence of nonunion. Our results suggest that NSM00158 represents a new potential compound to prevent the occurrence of nonunion by disrupting CtBP2 function and impairing the assembly of the CtBP2-p300-Runx2 transcriptional complex.

• The Journal of Korean Medicine
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• v.29 no.3
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• pp.88-99
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• 2008

Objectives: In order to identify the anti-inflammatory and analgesic properties of Salvia miltiorrhiza (Dan-Sam), widely used in Korean traditional medicine, an in vitro screening system was designed using pGL3, a luciferase reporter vector, and the tumor necrosis factor (TNF)-${\alpha}$ and cyclooxygenase (COX)-2 as target genes. Methods: The promoter regions of each gene were generated by PCR using the human chromosome as template DNA, and inserted into pGL3 vector with Kpn I and Hind III. The final construct was transfected into human myelomonocytic leukemia cells (U-937) that could be differentiated and activated by phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS). Using this system, the anti-inflammatory and analgesic effects of several herbal extracts regarded to have the medicinal effects of diminishing body heat and complementing Qi were tested. The chemicals PD98059 and berberine chloride were used as controls of the transcriptional inhibitors of TNF-${\alpha}$ and COX-2, respectively. Results: Salvia miltiorrhiza (Dan-Sam) demonstrated significant decrease of TNF-${\alpha}$ and COX-2 mRNA in the in vitro assay system. In MTT assay, Salvia miltiorrhiza (Dan-Sam) did not significantly inhibit the survival and proliferation of human myelomonocytic leukemia cells (U-937). Conclusions: Salvia miltiorrhiza (Dan-Sam) was found to exhibit the significant medicinal properties of anti-inflammatory and analgesic effects.

• Journal of the Korean Society of Food Science and Nutrition
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• v.14 no.2
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• pp.202-213
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• 1985

The protein nutritional quality of foods has become an important factor to food processors with the advent of nutritional labeling regulations for foods. Then, as is true today, the officially approved assay for protein nutritional quality was the rat based protein efficiency ratio(PER) bioassay. The PER bioassay requires a minimum of 28 days to performe, and is therefore not applicable to routine quality assurance use by the food industry. Within the past ten years there has been a research emphasis placed on the development of rapid, inexpensive, biological and/or chemical based assays for protein nutritional quality. It was hoped that if a rapid assay could be developed and thoroughly tested, it could be used in lieu of the PER bioassay in the day-to-day quality assurance screening of food ingredients and products. The rapid assays developed in the hope of attaining this goal have been based on microorganisms, proteolytic enzymes, and amino acid profiles, as well as combinations of the above. In this review, it will be described and briefly discussed many of procedures which had contributed conceptually as well as practically to the development of in vitro methods for the evaluation of protein quality. Special emphasis will be placed on the C-PER(computed protein efficiency ratio) assay which combines data from in vitro protease digestion and amino acid composition to predict protein nutritional quality designed by Satterlee et al. (1980), and the DC-PER(discriminant computed PER) which is a method of estimating protein quality based on rat assay and in vitro digestibility obtained using solely essential amino acid data will be also introduced.

• Bulletin of the Korean Chemical Society
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• v.31 no.11
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• pp.3313-3317
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• 2010

A simple and efficient synthetic route for making purpurin-18-N-aminoimides is described. The purpurin-18-Naminoimides are obtained by treatment of purpurin-18 methyl ester with various amines. These new compounds have long wavelength absorptions in the range of 706 - 711 nm. In preliminary screening, the purpurin-18-N-aminoimides have shown promising photosensitizing activity for the cancer cell by in vitro study in photodynamic therapy.