• Proceedings of the National Institute of Ecology of the Republic of Korea
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• v.2 no.3
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• pp.210-218
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• 2021

Recently, pest-resistant living modified (LM) crops developed using RNA interference (RNAi) technology have been imported into South Korea. However, the potential adverse effects of unintentionally released RNAi-based LM crops on non-target species have not yet been reported. Coccinella septempunctata, which feeds on aphids, is an important natural enemy insect which can be exposed to the double-stranded RNA (dsRNA) produced by RNAi-based LM plants. To assess the risk of ingestion of Snf7 dsRNA by C. septempunctata, we first identified the species through morphological analysis of collected insects. A method for species identification at the gene level was developed using a specific C. septempunctata 12S rRNA. Furthermore, an experimental model was devised to assess the risk of Snf7 dsRNA ingestion in C. septempunctata. Snf7 dsRNA was mass-purified using an effective dsRNA synthesis method and its presence in C. septempunctata was confirmed after treatment with purified Snf7 dsRNA. Finally, the survival rate, development time, and dry weight of Snf7 dsRNA-treated C. septempunctata were compared with those of GFP and vATPase A dsRNA control treatments, and no risk was found. This study illustrates an effective Snf7 dsRNA synthesis method, as well as a high-concentration domestic insect risk assessment method which uses dsRNA to assess the risk of unintentional released of LM organisms against non-target species.

• Animal Bioscience
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• v.36 no.2
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• pp.209-217
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• 2023

Objective: The conservation of Bali bulls, the Indonesian native breed of cattle, is crucial for cattle breeding in Indonesia. To guarantee the spread of Bali bulls through artificial insemination the quality of the frozen semen must be high. To this end, adding an extender material to semen that increases spermatozoa's survival during cryopreservation is important. Green tea extract (GTE) can be used as cryoprotectant because its high antioxidant activity can help avoid reactive oxygen species formation. Methods: Semen of five Bali bulls from the National Artificial Insemination Center at Singosari, Indonesia was collected routinely twice a week. First, fresh semen inspection was performed to determine the feasibility of using Bali bulls as animal samples. The extender used in this study was Tris-based egg yolk. The samples were divided into four treatments: T0, no GTE added to the extender; T1, 0.05 mg GTE plus 100 mL extender; T2, 0.10 mg GTE plus 100 mL extender; and T3, 0.15 mg GTE plus 100 mL extender. The semen freezing process was conducted according to standard procedures and sperm quality parameters, i.e., sperm motility, viability, abnormalities, and membrane integrity observed pre-freezing and post-thawing. Results: There were significant differences in total motility, progressive motility, viability, and integrity membrane of Bali bull sperm at both pre-freezing and post-thawing after adding GTE into the extender. In contrast, there were no differences in abnormalities among treatments. Conclusion: Adding GTE at a 0.15 mg into 100 mL Tris-based egg yolk extender can improve the quality of cryopreserved Bali bull sperm.

• Molecules and Cells
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• v.45 no.11
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• pp.846-854
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• 2022

Neurons make long-distance connections via their axons, and the accuracy and stability of these connections are crucial for brain function. Research using various animal models showed that the molecular and cellular mechanisms underlying the assembly and maintenance of neuronal circuitry are highly conserved in vertebrates. Therefore, to gain a deeper understanding of brain development and maintenance, an efficient vertebrate model is required, where the axons of a defined neuronal cell type can be genetically manipulated and selectively visualized in vivo. Placental mammals pose an experimental challenge, as time-consuming breeding of genetically modified animals is required due to their in utero development. Xenopus laevis, the most commonly used amphibian model, offers comparative advantages, since their embryos ex utero during which embryological manipulations can be performed. However, the tetraploidy of the X. laevis genome makes them not ideal for genetic studies. Here, we use Xenopus tropicalis, a diploid amphibian species, to visualize axonal pathfinding and degeneration of a single central nervous system neuronal cell type, the retinal ganglion cell (RGC). First, we show that RGC axons follow the developmental trajectory previously described in X. laevis with a slightly different timeline. Second, we demonstrate that co-electroporation of DNA and/or oligonucleotides enables the visualization of gene function-altered RGC axons in an intact brain. Finally, using this method, we show that the axon-autonomous, Sarm1-dependent axon destruction program operates in X. tropicalis. Taken together, the present study demonstrates that the visual system of X. tropicalis is a highly efficient model to identify new molecular mechanisms underlying axon guidance and survival.

• Biomolecules & Therapeutics
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• v.31 no.1
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• pp.116-126
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• 2023

Mainly due to the slanted focus on the mechanism and regulation of neuronal aging, research on astrocyte aging and its modulation during brain aging is scarce. In this study, we established aged astrocyte culture model by long-term culturing. Cellular senescence was confirmed through SA-β-gal staining as well as through the examination of morphological, molecular, and functional markers. RNA sequencing and functional analysis of astrocytes were performed to further investigate the detailed characteristics of the aged astrocyte model. Along with aged phenotypes, decreased astrocytic proliferation, migration, mitochondrial energetic function and support for neuronal survival and differentiation has been observed in aged astrocytes. In addition, increased expression of cytokines and chemokine-related factors including plasminogen activator inhibitor -1 (PAI-1) was observed in aged astrocytes. Using the RNA sequencing results, we searched potential drugs that can normalize the dysregulated gene expression pattern observed in long-term cultured aged astrocytes. Among several candidates, minoxidil, a pyrimidine-derived anti-hypertensive and anti-pattern hair loss drug, normalized the increased number of SA-β-gal positive cells and nuclear size in aged astrocytes. In addition, minoxidil restored up-regulated activity of PAI-1 and increased mitochondrial superoxide production in aged astrocytes. We concluded that long term culture of astrocytes can be used as a reliable model for the study of astrocyte senescence and minoxidil can be a plausible candidate for the regulation of brain aging.

• Biomedical Science Letters
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• v.28 no.4
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• pp.334-339
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• 2022

African swine fever virus (ASFV) is a highly contagious and lethal pathogen that poses a threat to the global pork industry. The World Organization for Animal Health (WOAH) has placed strict surveillance measures for ASFV. The possibility of long-term survival of ASFV in raw meat or undercooked pork has been reported. Accordingly, the problem of secondary infection in food waste from households or waste disposal facilities has emerged, raising the need for ASFV monitoring of food waste. However, most of the previously reported ASFV gene detection methods are focused on clinical monitoring of pigs. There are very few cases in which their application in waste has been verified. Since ASFV diagnosis requires rapid monitoring and immediate action, loop-mediated isothermal amplification (LAMP) may be suitable, but this requires conformity assessment for LAMP to be used as a diagnostic technique. In this study, six LAMP methods were evaluated, and two methods (kit and manual) were recommended for use in diagnosing ASFV in food waste.

• Journal of Digestive Cancer Research
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• v.1 no.1
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• pp.24-28
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• 2013

The five-year survival for patients with gastric cancer improved only modestly over the last 50 years. So, several studies about molecular target chemotherapy were investigated. We reviewed about molecular target chemotherapy for advanced unresectable and metastatic gastric cancer, which has developed recently. EGFR (Epidermal growth factor receptor), HER (Human epidermal growth factor receptor), VEGF (Vascular endothelial growth factor receptor) can be the target of therapy for gastric cancer. Patients with advanced gastric adenocarcinoma who are potential candidates for trastuzumab should have their tumors assayed for the presence of HER2 overexpression utilizing tumor-specific criteria and/or gene amplification. We suggest the addition of trastuzumab to chemotherapy in patients with HER2-positive tumors (as defined by 3+ immunohistochemical staining or FISH positivity), as long as they do not have a contraindication to trastuzumab. Except for trastuzumab, we summarized several studies for molecular target agents which were not validated yet.

• BMB Reports
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• v.56 no.11
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• pp.600-605
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• 2023

Intrahepatic cholangiocarcinoma (ICC) is a bile duct cancer and a rare malignant tumor with a poor prognosis owing to the lack of an early diagnosis and resistance to conventional chemotherapy. A combination of gemcitabine and cisplatin is the typically attempted first-line treatment approach. However, the underlying mechanism of resistance to chemotherapy is poorly understood. We addressed this by studying dynamics in the human ICC SCK cell line. Here, we report that the regulation of glucose and glutamine metabolism was a key factor in overcoming cisplatin resistance in SCK cells. RNA sequencing analysis revealed a high enrichment cell cycle-related gene set score in cisplatin-resistant SCK (SCK-R) cells compared to parental SCK (SCK WT) cells. Cell cycle progression correlates with increased nutrient requirement and cancer proliferation or metastasis. Commonly, cancer cells are dependent upon glucose and glutamine availability for survival and proliferation. Indeed, we observed the increased expression of GLUT (glucose transporter), ASCT2 (glutamine transporter), and cancer progression markers in SCK-R cells. Thus, we inhibited enhanced metabolic reprogramming in SCK-R cells through nutrient starvation. SCK-R cells were sensitized to cisplatin, especially under glucose starvation. Glutaminase-1 (GLS1), which is a mitochondrial enzyme involved in tumorigenesis and progression in cancer cells, was upregulated in SCK-R cells. Targeting GLS1 with the GLS1 inhibitor CB-839 (telaglenastat) effectively reduced the expression of cancer progression markers. Taken together, our study results suggest that a combination of GLUT inhibition, which mimics glucose starvation, and GLS1 inhibition could be a therapeutic strategy to increase the chemosensitivity of ICC.

• Biomolecules & Therapeutics
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• v.31 no.6
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• pp.629-639
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• 2023

Cardiovascular diseases (CVDs) are the most common cardiovascular system disorders. Cellular senescence is a key mechanism associated with dysfunction of aged vascular endothelium. Hyaluronic acid and proteoglycan link protein 1 (HAPLN1) has been known to non-covalently link hyaluronic acid (HA) and proteoglycans (PGs), and forms and stabilizes HAPLN1-containing aggregates as a major component of extracellular matrix. Our previous study showed that serum levels of HAPLN1 decrease with aging. Here, we found that the HAPLN1 gene expression was reduced in senescent human umbilical vein endothelial cells (HUVECs). Moreover, a recombinant human HAPLN1 (rhHAPLN1) decreased the activity of senescence-associated β-gal and inhibited the production of senescence-associated secretory phenotypes, including IL-1β, CCL2, and IL-6. rhHAPLN1 also downregulated IL-17A levels, which is known to play a key role in vascular endothelial senescence. In addition, rhHAPLN1 protected senescent HUVECs from oxidative stress by reducing cellular reactive oxygen species levels, thus promoting the function and survival of HUVECs and leading to cellular proliferation, migration, and angiogenesis. We also found that rhHAPLN1 not only increases the sirtuin 1 (SIRT1) levels, but also reduces the cellular senescence markers levels, such as p53, p21, and p16. Taken together, our data indicate that rhHAPLN1 delays or inhibits the endothelial senescence induced by various aging factors, such as replicative, IL-17A, and oxidative stress-induced senescence, thus suggesting that rhHAPLN1 may be a promising therapeutic for CVD and atherosclerosis.

• Journal of Marine Bioscience and Biotechnology
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• v.15 no.2
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• pp.33-40
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• 2023

This study aimed to investigate the immunomodulatory function of Pyropia yezoensis hydrothermal (water) extract (PYWE) in comparison to the group treated only with lipopolysaccharides (LPS) in RAW264.7 cells. LPS is known to be an inflammatory mediator that activates macrophages, leading to the secretion of nitric oxide (NO), inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) as defense responses. Through enzyme-linked immunoassay and western blot analyses, it was observed that PYWE increased the expression levels of NO, iNOS, TNF-α, and IL-6 in RAW264.7 cells in a dose-dependent manner, although to a lesser extent compared with the group treated with LPS alone. In addition, the study examined the mitogen-activated protein kinases (MAPKs) pathway, which regulates various cellular activities, including gene expression, mitosis, cell differentiation, transformation, survival, and death. The western blot analysis confirmed that PYWE also regulated the MAPKs pathway. Furthermore, the expression levels of immunomodulatory-related factors increased in the group treated with PYWE compared with the control group. Even though the effects of PYWE were usually less strong than those of LPS, the effects of PYWE increased with increasing doses compared to the control group. This suggests that PYWE could be used to control the immune system.

• Korean Circulation Journal
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• v.53 no.6
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• pp.351-366
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• 2023

Along with the development of immunosuppressive drugs, major advances on xenotransplantation were achieved by understanding the immunobiology of xenograft rejection. Most importantly, three predominant carbohydrate antigens on porcine endothelial cells were key elements provoking hyperacute rejection: α1,3-galactose, SDa blood group antigen, and N-glycolylneuraminic acid. Preformed antibodies binding to the porcine major xenoantigen causes complement activation and endothelial cell activation, leading to xenograft injury and intravascular thrombosis. Recent advances in genetic engineering enabled knock-outs of these major xenoantigens, thus producing xenografts with less hyperacute rejection rates. Another milestone in the history of xenotransplantation was the development of co-stimulation blockaded strategy. Unlike allotransplantation, xenotransplantation requires blockade of CD40-CD40L pathway to prevent T-cell dependent B-cell activation and antibody production. In 2010s, advanced genetic engineering of xenograft by inducing the expression of multiple human transgenes became available. So-called 'multi-gene' xenografts expressing human transgenes such as thrombomodulin and endothelial protein C receptor were introduced, which resulted in the reduction of thrombotic events and improvement of xenograft survival. Still, there are many limitations to clinical translation of cardiac xenotransplantation. Along with technical challenges, zoonotic infection and physiological discordances are major obstacles. Social barriers including healthcare costs also need to be addressed. Although there are several remaining obstacles to overcome, xenotransplantation would surely become the novel option for millions of patients with end-stage heart failure who have limited options to traditional therapeutics.