• Title/Summary/Keyword: Real-time polymerase reaction

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Alteration of MicroRNAs Targeted Integrins by PD-MSCs Transplantation Is Involved in Hepatic Regeneration in a Rat Model with BDL (담관결찰 쥐 모델에서 태반유래중간엽줄기세포 이식에 의한 miRNA 표적 인테그린 변화의 간재생 효과)

  • Park, Sohae
    • Journal of Life Science
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    • v.31 no.8
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    • pp.710-718
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    • 2021
  • Placenta-derived mesenchymal stem cells (PD-MSCs) are promising candidates for cell-based therapy in regenerative medicine. The migration and homing potential of PD-MSCs to injured sites is a critical property of MSC engraftment. MicroRNAs (miRNAs) have recently been shown to regulate the critical functions of MSCs, such as proliferation, survival, and migration. The objective of the present study was to identify the miRNA and target genes involved in PD-MSCs homing in a bile duct ligation (BDL) rat model. We selected candidate miRNAs targeting genes for PD-MSCs homing based on microarray analysis. PD-MSC engraftment in BDL-injured rat liver was identified by immunofluorescence assay and human-specific Alu gene expression by quantitative real-time polymerase chain reaction (qRT-PCR) one week after transplantation. Compared with migrated naïve PD-MSCs under hypoxic and normoxic conditions (Hyp/Nor), the transplanted group with PD-MSCs (Tx) showed distinct differences in miRNA expressions in BDL-injured rat liver. We also validated the miRNAs and their target genes for PD-MSCs homing. The expressions of integrin α4 (ITGA4) and integrin α5 (ITGA5) target genes for miR-199a-5p and miR-148a-3p were significantly upregulated in the Tx group (p<0.05). In addition, integrin β1 (ITGB1) and integrin β8 (ITGB8) were upregulated by suppressing miR-183-5p and miR-145-5p, respectively. These results demonstrated that PD-MSCs regulate miRNA expression related to the integrin family for their homing effects on the BDL-injured rat liver. The findings further suggest that miRNA-mediated regulation of the integrin family contributes to the therapeutic efficacy of PD-MSCs in the rat hepatic fibrosis model by BDL.

Inhibitory effect of water-soluble mulberry leaf extract on hepatic lipid accumulation in high-fat diet-fed rats via modulation of hepatic microRNA-221/222 expression and inflammation (고지방식이 급여 쥐에서 수용성 뽕나무 잎 추출물의 간 microRNA-221/222 발현 및 염증 조절을 통한 간 지질 축적억제 효과)

  • Lee, Mak-Soon;Kim, Cheamin;Ko, Hyunmi;Kim, Yangha
    • Journal of Nutrition and Health
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    • v.55 no.2
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    • pp.227-239
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    • 2022
  • Purpose: This study investigated the effects of water-soluble mulberry leaf extract (ME) on hepatic lipid accumulation in high-fat diet-fed rats via the regulation of hepatic microRNA (miR)-221/222 and inflammation. Methods: Male Sprague-Dawley rats (4 weeks old) were randomly divided into 3 groups (n = 7 each) and fed with 10 kcal% low-fat diet (LF), 45 kcal% high-fat diet (HF), or HF + 0.8% ME for 14 weeks. Lipid profiles and cytokine levels of the liver and serum were measured using commercial enzymatic colorimetric and enzyme-linked immunosorbent assay, respectively. The messenger RNA (mRNA) and miR levels in liver tissue were assayed by real-time quantitative reverse-transcription polymerase chain reaction. Results: Supplementation of ME reduces body weight and improves the liver and serum lipid profiles as compared to the HF group. The mRNA levels of hepatic peroxisome proliferator-activated receptor-gamma, sterol regulatory element binding protein-1c, fatty acid synthase, and fatty acid translocase, which are genes involved in lipid metabolism, were significantly downregulated in the ME group compared to the HF group. In contrast, the mRNA level of hepatic carnitine palmitoyl transferase-1 (involved in fatty acid oxidation) was upregulated by ME supplementation. Furthermore, administration of ME significantly downregulated the mRNA levels of inflammatory mediators such as hepatic tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), monocyte chemoattractant protein-1, and inducible nitric oxide synthase. The serum levels of TNF-α, IL-6, and nitric oxide were also significantly reduced in ME group compared to the HF group. Expression of hepatic miR-221 and miR-222, which increase in the inflammatory state of the liver, were also significantly inhibited in the ME group compared to the HF group. Conclusion: These results indicate that ME has the potential to improve hepatic lipid accumulation in high-fat diet-fed rats via modulation of inflammatory mediators and hepatic miR-221/222 expressions.

A study on the effect of microgroove-fibronectin complex titanium plate on the expression of various cell behavior-related genes in human gingival fibroblasts (인간치은섬유아세포의 다양한 세포행동 관련 유전자발현에 마이크로그루브-파이브로넥틴 복합 티타늄표면이 미치는 영향에 대한 연구)

  • Hwang, Yu Jeong;Lee, Won Joong;Leesungbok, Richard;Lee, Suk Won
    • Journal of Dental Rehabilitation and Applied Science
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    • v.38 no.3
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    • pp.150-161
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    • 2022
  • Purpose: To determine the effects of the microgroove-fibronectin complex surface on the expression of various genes related to cellular activity in human gingival fibroblasts. Materials and Methods: Smooth titanium specimens (NE0), acid-treated titanium specimens (E0), microgroove and acid-treated titanium specimens (E60/10), fibronectin-fixed smooth titanium specimens (NE0FN), acid-treated and fibronectin-immobilized titanium specimens (E0FN), and microgroove and acid-treated titanium specimens immobilized with fibronectin (E60/10FN) were prepared. Real-time polymerase chain reaction experiments were conducted on 44 genes related to cell behavior of human gingival fibroblasts. Results: Adhesion and proliferation of human gingival fibroblast on microgroove-fibronectin complex titanium were activated through four types of signaling pathway. Integrin α5, Integrin β1, Integrin β3, Talin-2, which belong to the focal adhesion pathway, AKT1, AKT2, NF-κB, which belong to the PI3K-AKT signaling pathway, MEK2, ERK1, ERK2, which belong to the MAPK signaling pathway, and Cyclin D1, CDK4, CDK6 genes belonging to the cell cycle signaling pathway were upregulated on the microgroove-fibronectin complex titanium surface (E60/10FN). Conclusion: The microgroove-fibronectin complex titanium surface can up-regulate various genes involved in cell behavior.

Effects of polygalacin D extracted from Platycodon grandiflorum on myoblast differentiation and muscle atrophy (길경에서 추출한 polygalacin D가 근원세포 분화 및 근위축에 미치는 영향)

  • Eun-Ju Song;Ji-Won Heo;Jee Hee Jang;Eonmi Kim;Yun Hee Jeong;Min Jung Kim;Sung-Eun Kim
    • Journal of Nutrition and Health
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    • v.56 no.6
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    • pp.602-614
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    • 2023
  • Purpose: The balance between synthesis and degradation of proteins plays a critical role in the maintenance of skeletal muscle mass. Mitochondrial dysfunction has been closely associated with skeletal muscle atrophy caused by aging, cancer, and chemotherapy. Polygalacin D is a saponin derivative isolated from Platycodon grandiflorum (Jacq.) A. DC. This study aimed to investigate the effects of polygalacin D on myoblast differentiation and muscle atrophy in association with mitochondrial function in in vitro and in zebrafish models in vivo. Methods: C2C12 myoblasts were cultured in differentiation media containing different concentrations of polygalacin D, followed by the immunostaining of the myotubes with myosin heavy chain (MHC). The mRNA expression of markers related to myogenesis, muscle atrophy, and mitochondrial function was determined by real-time quantitative reverse transcription polymerase chain reaction. Wild type AB* zebrafish (Danio rerio) embryos were treated with 5-fluorouracil, leucovorin, and irinotecan (FOLFIRI) with or without polygalacin D, and immunostained to detect slow and fast types of muscle fibers. The Tg(Xla.Eef1a1:mitoEGFP) zebrafish expressing mitochondria-targeted green fluorescent protein was used to monitor mitochondrial morphology. Results: The exposure of C2C12 myotubes to 0.1 ng/mL of polygalacin D increased the formation of MHC-positive multinucleated myotubes (≥ 8 nuclei) compared with the control. Polygalacin D significantly increased the expression of MHC isoforms (Myh1, Myh2, Myh4, and Myh7) involved in myoblast differentiation while it decreased the expression of atrophic markers including muscle RING-finger protein-1 (MuRF1), mothers against decapentaplegic homolog (Smad)2, and Smad3. In addition, polygalacin D promoted peroxisome proliferator-activated receptor-gamma coactivator (Pgc1α) expression and reduced the level of mitochondrial fission regulators such as dynamin-1-like protein (Drp1) and mitochondrial fission 1 (Fis1). In a zebrafish model of FOLFIRI-induced muscle atrophy, polygalacin D improved not only mitochondrial dysfunction but also slow and fast muscle fiber atrophy. Conclusion: These results demonstrated that polygalacin D promotes myogenesis and alleviates chemotherapy-induced muscle atrophy by improving mitochondrial function. Thus, polygalacin D could be useful as nutrition support to prevent and ameliorate muscle wasting and weakness.

Effect of Oil in Water Nanoemulsion Containing a Mixture of Lactic Acid and Gluconolactone for Skin Barrier Improvement (유산 및 글루코노락톤 혼합물을 함유하는 수중유형 나노에멀젼의 피부장벽개선 효과)

  • Ji-Hye Hong;Young Duck Choi;Gye Won Lee;Young Ho Cho
    • Journal of Life Science
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    • v.33 no.11
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    • pp.905-914
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    • 2023
  • To evaluate the effectiveness of the skin barrier improvement of lactic acid (LA) and gluconolactone (GL), the expression of filaggrin, loricrin, hyaluronic acid (HA), hyaluronan syhthase-2 (HAS2), and aquaporine-3 (AQP3) in keratinocytes, and the moisture content and transepidermal water loss (TEWL) by clinical trials were evaluated. The expression levels of filaggrin and locricrin, which are the main factors affecting the proper functioning of skin barrier function, and HA, HAS2, and AQP3, which are skin moisturizing-related proteins measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. The results showed that the expression levels of the factors that decreased by H2O2 treatment were significantly increased by LA, GL, and a mixture of LA and GL at the mRNA and protein levels (p<0.05). The nanoemulsion containing a mixture of LA and GL was prepared using the emulsion inversion method, and the average particle size was 299.9 ± 0.287 nm. After measuring the TEWL of nanoemulsion using Vapometer, it was found that TEWL significantly decreased by 15.53% and 26.73% after two weeks and four weeks of product use, respectively, compared to TEWL before product use (p<0.001). Similarly, the skin moisture content of the nanoemulsion significantly increased by 15.40% and 26.59% after two weeks and four weeks of product use, respectively, compared to skin moisture content before product use (p<0.001). Therefore, the skin barrier function and moisturizing effect of a mixture of LA and GL are shown by increasing the moisture content and decreasing the TEWL by increasing the expression of filaggrin, loricrin, HA, HAS2, and AQP3. This suggests the possibility for the development of functional cosmetic ingredients in the future.

Field Studios of In-situ Aerobic Cometabolism of Chlorinated Aliphatic Hydrocarbons

  • Semprini, Lewts
    • Proceedings of the Korean Society of Soil and Groundwater Environment Conference
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    • 2004.04a
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    • pp.3-4
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    • 2004
  • Results will be presented from two field studies that evaluated the in-situ treatment of chlorinated aliphatic hydrocarbons (CAHs) using aerobic cometabolism. In the first study, a cometabolic air sparging (CAS) demonstration was conducted at McClellan Air Force Base (AFB), California, to treat chlorinated aliphatic hydrocarbons (CAHs) in groundwater using propane as the cometabolic substrate. A propane-biostimulated zone was sparged with a propane/air mixture and a control zone was sparged with air alone. Propane-utilizers were effectively stimulated in the saturated zone with repeated intermediate sparging of propane and air. Propane delivery, however, was not uniform, with propane mainly observed in down-gradient observation wells. Trichloroethene (TCE), cis-1, 2-dichloroethene (c-DCE), and dissolved oxygen (DO) concentration levels decreased in proportion with propane usage, with c-DCE decreasing more rapidly than TCE. The more rapid removal of c-DCE indicated biotransformation and not just physical removal by stripping. Propane utilization rates and rates of CAH removal slowed after three to four months of repeated propane additions, which coincided with tile depletion of nitrogen (as nitrate). Ammonia was then added to the propane/air mixture as a nitrogen source. After a six-month period between propane additions, rapid propane-utilization was observed. Nitrate was present due to groundwater flow into the treatment zone and/or by the oxidation of tile previously injected ammonia. In the propane-stimulated zone, c-DCE concentrations decreased below tile detection limit (1 $\mu$g/L), and TCE concentrations ranged from less than 5 $\mu$g/L to 30 $\mu$g/L, representing removals of 90 to 97%. In the air sparged control zone, TCE was removed at only two monitoring locations nearest the sparge-well, to concentrations of 15 $\mu$g/L and 60 $\mu$g/L. The responses indicate that stripping as well as biological treatment were responsible for the removal of contaminants in the biostimulated zone, with biostimulation enhancing removals to lower contaminant levels. As part of that study bacterial population shifts that occurred in the groundwater during CAS and air sparging control were evaluated by length heterogeneity polymerase chain reaction (LH-PCR) fragment analysis. The results showed that an organism(5) that had a fragment size of 385 base pairs (385 bp) was positively correlated with propane removal rates. The 385 bp fragment consisted of up to 83% of the total fragments in the analysis when propane removal rates peaked. A 16S rRNA clone library made from the bacteria sampled in propane sparged groundwater included clones of a TM7 division bacterium that had a 385bp LH-PCR fragment; no other bacterial species with this fragment size were detected. Both propane removal rates and the 385bp LH-PCR fragment decreased as nitrate levels in the groundwater decreased. In the second study the potential for bioaugmentation of a butane culture was evaluated in a series of field tests conducted at the Moffett Field Air Station in California. A butane-utilizing mixed culture that was effective in transforming 1, 1-dichloroethene (1, 1-DCE), 1, 1, 1-trichloroethane (1, 1, 1-TCA), and 1, 1-dichloroethane (1, 1-DCA) was added to the saturated zone at the test site. This mixture of contaminants was evaluated since they are often present as together as the result of 1, 1, 1-TCA contamination and the abiotic and biotic transformation of 1, 1, 1-TCA to 1, 1-DCE and 1, 1-DCA. Model simulations were performed prior to the initiation of the field study. The simulations were performed with a transport code that included processes for in-situ cometabolism, including microbial growth and decay, substrate and oxygen utilization, and the cometabolism of dual contaminants (1, 1-DCE and 1, 1, 1-TCA). Based on the results of detailed kinetic studies with the culture, cometabolic transformation kinetics were incorporated that butane mixed-inhibition on 1, 1-DCE and 1, 1, 1-TCA transformation, and competitive inhibition of 1, 1-DCE and 1, 1, 1-TCA on butane utilization. A transformation capacity term was also included in the model formation that results in cell loss due to contaminant transformation. Parameters for the model simulations were determined independently in kinetic studies with the butane-utilizing culture and through batch microcosm tests with groundwater and aquifer solids from the field test zone with the butane-utilizing culture added. In microcosm tests, the model simulated well the repetitive utilization of butane and cometabolism of 1.1, 1-TCA and 1, 1-DCE, as well as the transformation of 1, 1-DCE as it was repeatedly transformed at increased aqueous concentrations. Model simulations were then performed under the transport conditions of the field test to explore the effects of the bioaugmentation dose and the response of the system to tile biostimulation with alternating pulses of dissolved butane and oxygen in the presence of 1, 1-DCE (50 $\mu$g/L) and 1, 1, 1-TCA (250 $\mu$g/L). A uniform aquifer bioaugmentation dose of 0.5 mg/L of cells resulted in complete utilization of the butane 2-meters downgradient of the injection well within 200-hrs of bioaugmentation and butane addition. 1, 1-DCE was much more rapidly transformed than 1, 1, 1-TCA, and efficient 1, 1, 1-TCA removal occurred only after 1, 1-DCE and butane were decreased in concentration. The simulations demonstrated the strong inhibition of both 1, 1-DCE and butane on 1, 1, 1-TCA transformation, and the more rapid 1, 1-DCE transformation kinetics. Results of tile field demonstration indicated that bioaugmentation was successfully implemented; however it was difficult to maintain effective treatment for long periods of time (50 days or more). The demonstration showed that the bioaugmented experimental leg effectively transformed 1, 1-DCE and 1, 1-DCA, and was somewhat effective in transforming 1, 1, 1-TCA. The indigenous experimental leg treated in the same way as the bioaugmented leg was much less effective in treating the contaminant mixture. The best operating performance was achieved in the bioaugmented leg with about over 90%, 80%, 60 % removal for 1, 1-DCE, 1, 1-DCA, and 1, 1, 1-TCA, respectively. Molecular methods were used to track and enumerate the bioaugmented culture in the test zone. Real Time PCR analysis was used to on enumerate the bioaugmented culture. The results show higher numbers of the bioaugmented microorganisms were present in the treatment zone groundwater when the contaminants were being effective transformed. A decrease in these numbers was associated with a reduction in treatment performance. The results of the field tests indicated that although bioaugmentation can be successfully implemented, competition for the growth substrate (butane) by the indigenous microorganisms likely lead to the decrease in long-term performance.

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