• Title/Summary/Keyword: RT-qPCR

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LC-MS Analysis According to the Combined Treatment of Paenibacillus yonginensis DCY84T and Silicon in Rice

  • Yo-Han Yoo;Mee Youn Lee;Yeon-Ju Kim;Eok-Keun Ahn;Ki-Hong Jung
    • Proceedings of the Korean Society of Crop Science Conference
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    • 2022.10a
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    • pp.232-232
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    • 2022
  • We reported in our recent studies that the combined treatment of Paenibacillus yonginensis DCY84T (DCY84T) and Silicon (Si) promotes initial plant growth and increases resistance to biotic and abiotic stress. To understand the molecular background of these phenotypes, Liquid Chromatography Mass Spectrometry (LC-MS) analysis was performed, and it was confirmed that unsaturated fatty acid metabolites such as oleic acid and linoleic acid decreased in response to the combined treatment of DCY84T and Si. The stearoyl-acyl carrier protein desaturase (SACPD) introduces the cis double bond into the acyl-ACPs at C9, resulting in the production of unsaturated fatty acid. We identified OsSSI2 encoding SACPD in rice and found that the expression of OsSSI2 was reduced under DCY84T and Si treatment. Furthermore, qRT-PCR analysis revealed that the expression of OsWRKY45, which is downstream of OsSSI2, was upregulated in response to DCY84T and Si treatment. These results enable the speculation that activation of the salicylic acid (SA)-responsive gene, OsWRKY45, may contribute to enhancing biological stress resistance. Based on this, we propose a probable model for the rice defense pathway following DCY84T and Si treatment. This model retains a WRKY45-dependent but NH1(NPR1)-independent SA signaling pathway.

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Comparison of Gene Expression Changes in Three Wheat Varieties with Different Susceptibilities to Heat Stress Using RNA-Seq Analysis

  • Myoung Hui Lee;Kyeong-Min Kim;Wan-Gyu Sang;Chon-Sik Kang;Changhyun Choi
    • Proceedings of the Korean Society of Crop Science Conference
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    • 2022.10a
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    • pp.197-197
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    • 2022
  • Wheat is highly susceptible to heat stress, which significantly reduces grain yield. In this study, we used RNA-seq technology to analyze the transcript expression at three different time-points after heat treatment in three cultivars differing in their susceptibility to heat stress: Jopum, Keumkang, and Olgeuru. A total of 11,751, 8850, and 14,711; 10,959,7946, and 14,205; and 22,895,13,060, and 19,408 differentially-expressed genes (log2 fold-change > 1 and FDR (padj) < 0.05) were identified in Jopum, Keumkang, and Olgeuru in the control vs. 6-h, in the control vs. 12-h, and in the 6-h vs. 12-h heat treatment, respectively. Functional enrichment analysis showed that the biological processes for DEGs, such as the cellular response to heat and oxidative stress-and including the removal of superoxide radicals and the positive regulation of superoxide dismutase activity-were significantly enriched among the three comparisons in all three cultivars. Furthermore, we investigated the differential expression patterns of reactive oxygen species (ROS)-scavenging enzymes, heat shock proteins, and heat-stress transcription factors using qRT-PCR to confirm the differences in gene expression among the three varieties under heat stress. This study contributes to a better understanding of the wheat heat-stress response at the early growth stage and the varietal differences in heat tolerancea.

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The Effect of Scalp Acupuncture and rTMS on Neuromotor Function in Photothrombotic Stroke Rat Model

  • Jong-Seong Park;Eun-Jong Kim;Min-Keun Song;Jung-Kook Kim;Ganbold Selenge;Sam-Gyu Lee
    • Biomedical Science Letters
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    • v.29 no.4
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    • pp.263-273
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    • 2023
  • This study aimed to investigate effect of scalp acupuncture (SA) and repetitive transcranial magnetic stimulation (rTMS) intervention on neuromotor function in photothrombotic cerebral infarction (PCI) rat model. Sixty male SD rats were used. PCI was induced on M1 cortex of right frontal lobe. SA was performed at the Qianding (GV21), Xuanli (GB6) acupoints of ipsilesional M1. Low-frequency rTMS was delivered to contralesional M1. All rats were randomly divided into 4 groups: group A, normal (n, 15); group B, PCI without any stimulation intervention (n, 15); group C, PCI with SA (n, 15); group D, PCI with rTMS (n, 15). Rota-rod test and Ladder rung walking test (LWT) were done weekly for 8 weeks after PCI. SA or rTMS was started from post-PCI 4th day as protocol for 8 weeks. H/E stain and IHC were done. Western blot and qRT-PCR study were performed for MAP2 and BDNF from ipsilesional M1 peri-infarction tissue. Brain MRI study was conducted to quantify the volume of cerebral infarction. As a result, left forelimb and hindlimb function significantly improved more in group C and D than control group, with expressed more BDNF and MAP2. And brain MRI showed focal infarction of right M1 after PCI, and infarction volume progressively decreased in group C and D than group B from post-PCI 5th to 8th week. SA or rTMS was more effective than no intervention group on neuromotor function of PCI rat model. The functional recovery was associated with stimulation intervention-related neurogenesis.

Inhibitory Effects of The Flower from Abeliophyllum distichum cv. Okhwang 1 on Melanogenesis in B16 F10 Cells

  • Mi-Ji Noh;Hye-Jeong Park;So-Yeon Han;Jeong-Yong Park;Seo-Hyun Yun;Soo-Yeon Kim;Tae-Won Jang;Jae-Ho Park
    • Proceedings of the Plant Resources Society of Korea Conference
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    • 2021.04a
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    • pp.53-53
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    • 2021
  • Abeliophyllum distichum Nakai (A. distichum), endemic species of Korea, is classified according to the petals and calyx colors. Recently, A. distichum cv. Okhwang 1, which has the golden flower, designated the first official cultivar improved from A. distichum species. The study on the chloroplast genome of A. distichum cv. Okhwang 1 have been reported, but no studies on bioactivity such as antioxidant, anti-inflammatory, and anti-cancer have been progressed. This study was conducted to evaluate the inhibition on melanogenesis of the flower from A. distichum cv. Okhwang 1 (FAO). Antioxidant activity was measured using DPPH and ABTS radical scavenging assays. Inhibition effects on melanogenesis of FAO were confirmed by expression of tyrosinase-related proteins and mRNAs using immunoblotting and RT-qPCR. Tyrosinase is an enzyme that regulates both stimulation and inhibition of melanogenesis. Stimulated MITF in cellular levels increases the expressions of tyrosinase, TRP-1, and TRP-2 to induce melanogenesis. As a result, FAO inhibited the expression of MITF, followed by down-regulated tyrosinase, TRP-1, and TRP-2, which lead to inhibit melanin overproduction. In conclusion, these results indicated that FAO reduced reactive oxygen species (ROS) and markedly inhibited the expression of melanin-related factors. The present study suggested providing that FAO has the potential for development as a functional cosmetic material derived from natural.

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Limited in vitro differentiation of porcine induced pluripotent stem cells into endothelial cells

  • In-Won Lee;Hyeon-Geun Lee;Dae-Ky Moon;Yeon-Ji Lee;Bo-Gyeong Seo;Sang-Ki Baek;Tae-Suk Kim;Cheol Hwangbo;Joon-Hee Lee
    • Journal of Animal Reproduction and Biotechnology
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    • v.38 no.3
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    • pp.109-120
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    • 2023
  • Background: Pluripotent stem cells (PSCs) including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer the immense therapeutic potential in stem cell-based therapy of degenerative disorders. However, clinical trials of human ESCs cause heavy ethical concerns. With the derivation of iPSCs established by reprogramming from adult somatic cells through the transgenic expression of transcription factors, this problems would be able to overcome. In the present study, we tried to differentiate porcine iPSCs (piPSCs) into endothelial cells (ECs) for stem cell-based therapy of vascular diseases. Methods: piPSCs (OSKMNL) were induced to differentiation into ECs in four differentiation media (APEL-2, APEL-2 + 50 ng/mL of VEGF, EBM-2, EBM-2 + 50 ng/mL of VEGF) on cultured plates coated with matrigel® (1:40 dilution with DMEM/F-12 medium) for 8 days. Differentiation efficiency of these cells were exanimated using qRT-PCR, Immunocytochemistry, Western blotting and FACS. Results: As results, expressions of pluripotency-associated markers (OCT-3/4, SOX2 and NANOG) were higher observed in all porcine differentiated cells derived from piPSCs (OSKMNL) cultured in four differentiation media than piPSCs as the control, whereas endothelial-associated marker (CD-31) in the differentiated cells was not expressed. Conclusions: It can be seen that piPSCs (OSKMNL) were not suitable to differentiate into ECs in the four differentiation media unlike porcine epiblast stem cells (pEpiSCs). Therefore, it would be required to establish a suitable PSCs for differentiating into ECs for the treatment of cardiovascular diseases.

The characteristics of bovine satellite cells with highly scored genomic estimated breeding value

  • Jae Ho Han;Ji Suk Yu;Do Hyun Kim;Hyun Woo Choi
    • Journal of Animal Reproduction and Biotechnology
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    • v.38 no.3
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    • pp.177-187
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    • 2023
  • Background: The grading of Hanwoo (Korean native cattle) is based on four economic traits, and efforts have been continuously made to improve the genetic traits associated with these traits. There is a technology to predict the expected grade based on the 4 economic genetic SNP characteristics of Korean cattle calves using single nucleotide polymorphism (SNP) technology. Selection of highly proliferative, self-renewing, and differentiating satellite cells from cattle is a key technology in the cultured meat industry. Methods: We selected the Hanwoo with high and low-scored of genomic estimated breeding value (GEBV) by using the Hanwoo 50K SNP bead chip. We then isolated the bovine satellite cells from the chuck mass. We then conducted comparative analyses of cell proliferation, immunocytochemistry, qRT-PCR at short- and long-term culture. We also analyzed the differentiation capability at short term culture. Results: Our result showed that the proliferation was significantly high at High scored GEBV (Hs-GEBV) compared to Low scored GEBV (Ls-GEBV) at short- and long-term culture. The expression levels of Pax3 were significantly higher in Hs-GEBV bovine satellite cells at long-term culture. However, there were no significant differences in the expression levels of Pax7 between Hs- and Ls-GEBV bovine satellite cells at short- and long- term culture. The expression levels of MyoG and MyHC were significantly high at Ls-GEBV bovine satellite cells. Conclusions: Our results indicated that selection of bovine satellite cells by Hanwoo 50K SNP bead chip could be effective selection methods for massive producing of satellite cells.

Whitening effects of fermented Trigonotis radicans var. sericea with Lactobacillus brevis in α-MSH-stimulated B16F10 melanoma cells

  • Da-Eun Jeong;Byung-Oh Kim;Young-Je Cho
    • Food Science and Preservation
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    • v.31 no.2
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    • pp.227-234
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    • 2024
  • This study was designed to compare the whitening effects of 60% ethanol extracts of Trigonotis radicans var. sericea (TR) and Lactobacillus brevis-fermented T. radicans var. sericea (FTR). Measurement of cytotoxicity in B16-F10 melanoma cells to confirm the whitening effect, FTR showed higher cell viability than TR. FTR showed inhibitory activity on melanin contents similar to the normal group at concentrations of 50 and 100 ㎍/mL. MITF expression was used to confirm the effect on melanogenesis-related protein expression. TR and FTR showed significant concentration-dependent decrease, and FTR showed lower expressions than the normal group at concentrations of 25, 50, and 100 ㎍/mL. Additionally, the mRNA expression of melanogenesis-related genes (MC1R, Rab27a, TGF-β1 and Myo5a) were measured by RT-qPCR to confirm the whitening effect. In MC1R expression at a concentration of 100 ㎍/mL in FTR showed effective inhibitory activities, and in TGF-β1 expression, TR and FTR both showed effective activities compared to normal groups even at low concentrations. Results of myo5a and Rab27a, a similar pattern was shown, and FTR showed effective inhibitory activities at a concentration of 100 ㎍/mL. As a result, FTR had higher whitening effects through bioconversion and is expected to be a good material for whitening functional cosmetics.

Phenotypic characterization of pre-harvest sprouting resistance mutants generated by the CRISPR/Cas9-geminiviral replicon system in rice

  • Jong Hee Kim;Jihyeon Yu;Jin Young Kim;Yong Jin Park;Sangsu Bae;Kwon Kyoo Kang;Yu Jin Jung
    • BMB Reports
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    • v.57 no.2
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    • pp.79-85
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    • 2024
  • Pre-harvest sprouting is a critical phenomenon involving germination of seeds in the mother plant before harvest under relative humid conditions and reduced dormancy. In this paper, we generated HDR mutant lines with one region SNP (C/T) and an insertion of 6 bp (GGT/GGTGGCGGC) in OsERF1 genes for pre-harvest sprouting (PHS) resistance using CRISPR/Cas9 and a geminiviral replicon system. The incidence of HDR was 2.6% in transformed calli. T1 seeds were harvested from 12 HDR-induced calli and named ERF1-hdr line. Molecular stability, key agronomic properties, physiological properties, and biochemical properties of target genes in the ERF1-hdr line were investigated for three years. The ERF1-hdr line showed significantly enhanced seed dormancy and pre-harvest sprouting resistance. qRT-PCR analysis suggested that enhanced ABA signaling resulted in a stronger phenotype of PHS resistance. These results indicate that efficient HDR can be achieved through SNP/InDel replacement using a single and modular configuration applicable to different rice targets and other crops. This work demonstrates the potential to replace all genes with elite alleles within one generation and greatly expands our ability to improve agriculturally important traits.

Methanol extract of Elsholtzia fruticosa promotes 3T3-L1 preadipocyte differentiation

  • Deumaya Shrestha;Eunbin Kim;Krishna K. Shrestha;Sung-Suk Suh;Sung-Hak Kim;Jong Bae Seo
    • Journal of Animal Science and Technology
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    • v.66 no.1
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    • pp.204-218
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    • 2024
  • Elsholtzia fruticosa (EF) is present in tropical regions throughout South Asian countries as well as the Himalayas. Although it has been used as a traditional medicine to treat digestive, respiratory, and inflammatory issues, its effect on preadipocyte differentiation is unknown. In this study, we examined the effects of a methanol extract prepared from EF on the differentiation of 3T3-L1 preadipocytes. Cell differentiation was assessed by microscopic observation and oil-red O staining. The expression of adipogenic and lipogenic genes, including PPARγ and C/EBPα, was measured by western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR), to provide insight into adipogenesis and lipogenesis mechanisms. The results indicated that EF promotes the differentiation of 3T3-L1 preadipocytes, with elevated lipid accumulation occurring in a concentration-dependent manner without apparent cytotoxicity. EF enhances the expression of adipogenic and lipogenic genes, including PPARγ, FABP4, adiponectin, and FAS, at the mRNA and protein levels. The effect of EF was more pronounced during the early and middle stages of 3T3-L1 cell differentiation. Treatment with EF decreased C/EBP homologous protein (CHOP) mRNA and protein levels, while increasing C/EBPα and PPARγ expression. Treatment with EF resulted in the upregulation of cyclin E and CDK2 gene expression within 24 h, followed by a decrease at 48 h, demonstrating the early-stage impact of EF. A concomitant increase in cyclin-D1 levels was observed compared with untreated cells, indicating that EF modulates lipogenic and adipogenic genes through intricate mechanisms involving CHOP and cell cycle pathways. In summary, EF induces the differentiation of 3T3-L1 preadipocytes by increasing the expression of adipogenic and lipogenic genes, possibly through CHOP and cell cycle-dependent mechanisms.

Effects of bioflocs on immune responses of Fleshy shrimp, Fenneropenaeus chinensis postlarvae and adults as related to the different feeding abilities

  • Su-Kyoung Kim;Su Kyoung Kim;In-Kwon Jang;Je-Cheon Jun
    • Fisheries and Aquatic Sciences
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    • v.26 no.11
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    • pp.649-659
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    • 2023
  • The present study compared the structure of mixilliped of Fenneropenaeus chinensis between the larval and adult stage and investigated the effect of the structural difference on the immunity of F. chinensis. A fourteen day and a one-month long culture trial were conducted each with postlarvae and adults of F. chinensis in the biofloc, mixed water (50% biofloc:50% clear seawater) and seawater control. Immune-related genes mRNA expressions of postlarvae was analysed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). And the analysis of adult stage immunity was carried out using phenoloxidase (PO) enzyme activation in haemocyte. In the postlarvae, the final body weights were 51.43 and 58.47 mg for the biofloc water and the control seawater, respectively. On the other hand, the final body weights of the adults were significantly different between biofloc water and seawater. The survival rate showed the opposite trend to the growth rate. Immune related genes mRNA expression levels in the postlarvae in the biofloc water were significantly lower than those in the seawater. While, the adult stage showed significantly higher PO enzyme activations in the biofloc water than in the seawater with the PO enzyme activation increasing proportionally to the biofloc concentration. This result is considerably explained by the observations of setal morphological structures of the third maxilliped: postlarvae have short serrulated setae that compose the small 'net' structure while adults had long and dense plumose setae. It is understood that the morphological difference of the maxilliped structure resulted in the different feeding abilities in the postlarvae and the adult F. chinensis to use bioflocs as food source.