• Title/Summary/Keyword: fluorescence microscopy

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Transfer of Isolated Mitochondria to Bovine Oocytes by Microinjection (미세주입을 이용한 난자로의 분리된 미토콘드리아 전달)

  • Baek, Sang-Ki;Byun, June-Ho;Kim, Bo Gyu;Lee, A ram;Cho, Young-Soo;Kim, Ik-Sung;Seo, Gang-Mi;Chung, Se-Kyo;Lee, Joon-Hee;Woo, Dong Kyun
    • Journal of Life Science
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    • v.27 no.12
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    • pp.1445-1451
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    • 2017
  • Mitochondria play a central role in energy generation by using electron transport coupled with oxidative phosphorylation. They also participate in other important cellular functions including metabolism, apoptosis, signaling, and reactive oxygen species production. Therefore, mitochondrial dysfunction is known to contribute to a variety of human diseases. Furthermore, there are various inherited diseases of energy metabolism due to mitochondrial DNA (mtDNA) mutations. Unfortunately, therapeutic options for these inherited mtDNA diseases are extremely limited. In this regard, mitochondrial replacement techniques are taking on increased importance in developing a clinical approach to inherited mtDNA diseases. In this study, green fluorescence protein (GFP)-tagged mitochondria were isolated by differential centrifugation from a mammalian cell line. Using microinjection technique, the isolated GFP-tagged mitochondria were then transferred to bovine oocytes that were triggered for early development. During the early developmental period from bovine oocytes to blastocysts, the transferred mitochondria were observed using fluorescent microscopy. The microinjected mitochondria were dispersed rapidly into the cytoplasm of oocytes and were passed down to subsequent cells of 2-cell, 4-cell, 8-cell, morula, and blastocyst stages. Together, these results demonstrate a successful in vitro transfer of isolated mitochondria to oocytes and provide a model for mitochondrial replacement implicated in inherited mtDNA diseases and animal cloning.

Isolation and Morphological Identification of Fresh Water Green Algae from Organic Farming Habitats in Korea (유기농업 생태계로부터 담수 녹조류 분리 및 형태적 동정)

  • Kim, Min-Jeong;Shim, Chang-Ki;Kim, Yong-Ki;Hong, Sung-Jun;Park, Jong-Ho;Han, Eun-Jung;Jee, Hyeong-Jin;Yun, Jong-Chul;Kim, Suk-Chul
    • Korean Journal of Organic Agriculture
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    • v.22 no.4
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    • pp.743-760
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    • 2014
  • This study aimed to isolate and identify freshwater algae from the organic agricultural ecosystems and investigate its biological characteristics to study the possibility of utilizing a biomass freshwater algae in organic farming. In the survey area, average water temperature was $12.4{\sim}28.2^{\circ}C$ and the pH ranges were from 6.1 to 8.5. The solid culture method is more suitable than liquid culture method for isolation of freshwater algae with lower contamination level and higher isolation frequency. A total of 115 strains were isolated from six freshwater algae habitats in nine regions in Korea. BGMM (BG11 Modified Medium) amended with NaNO3 and $KNO_3$ as a nitrogen, and $Na_2CO_3$ as carbon source was designed to isolate and culture freshwater algae. Absorbance of freshwater algae culture has increased dramatically to four days and decreased after eight days after inoculation. CHK008 of the seven isolates showed the highest absorbance in seven days after culturing in BGMM. The optimal pH of BGMM for culturing freshwater algae was pH 6-7. As light intensity increased, growth of freshwater algae increased. Among the five kinds of carbon sources, glucose and galactose promoted good growth of freshwater algae in BGMM. The colony color of purified 16 green algae isolates showed a separation of green, dark and light green, and of them, eleven algae strains showed a strong fluorescent light under fluorescence microscopy. Cell size of the green algae showed a wide range of variation depending on the species. General morphology of the green algae strains was spherical. Chlamydomonas sp. was elliptical, and Chlorella sorokiniana was ellipsoidal and cylindrical. All strains of the green algae except for Chlamydomonas sp. did not have flagella. One isolate of Chlamydomonas sp. and five isolates of C. sorokiniana secreted mucus. Sixteen isolates of 16 green algae were identified as two family and six species, Chlorella vulgalis, C. sorokiniana, C. pyrenoidosa, C. kessleri, C. emersonii, and Chlamydomonas sp. based on their morphological characteristics.

Development of High Intensity Focused Ultrasound (HIFU) Mediated AuNP-liposomal Nanomedicine and Evaluation with PET Imaging

  • Ji Yoon Kim;Un Chul Shin;Ji Yong Park;Ran Ji Yoo;Soeku Bae;Tae Hyeon Choi;Kyuwan Kim;Young Chan Ann;Jin Sil Kim;Yu Jin Shin;Hokyu Lee;Yong Jin Lee;Kyo Chul Lee;Suhng Wook Kim;Yun-Sang Lee
    • Journal of Radiopharmaceuticals and Molecular Probes
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    • v.9 no.1
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    • pp.9-16
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    • 2023
  • Liposomes as drug delivery system have proved useful carrier for various disease, including cancer. In addition, perfluorocarbon cored microbubbles are utilized in conjunction with high-intensity focused-ultrasound (HIFU) to enable simultaneous diagnosis and treatment. However, microbubbles generally exhibit lower drug loading efficiency, so the need for the development of a novel liposome-based drug delivery material that can efficiently load and deliver drugs to targeted areas via HIFU. This study aims to develop a liposome-based drug delivery material by introducing a substance that can burst liposomes using ultrasound energy and confirm the ability to target tumors using PET imaging. Liposomes (Lipo-DOX, Lipo-DOX-Au, Lipo-DOX-Au-RGD) were synthesized with gold nanoparticles using an avidin-biotin bond, and doxorubicin was mounted inside by pH gradient method. The size distribution was measured by DLS, and encapsulation efficiency of doxorubicin was analyzed by UV-vis spectrometer. The target specificity and cytotoxicity of liposomes were assessed in vitro by glioblastoma U87mg cells to HIFU treatment and analyzed using CCK-8 assay, and fluorescence microscopy at 6-hour intervals for up to 24 hours. For the in vivo study, U87mg model mouse were injected intravenously with 1.48 MBq of 64Cu-labeled Lipo-DOX-Au and Lipo-DOX-Au-RGD, and PET images were taken at 0, 2, 4, 8, and 24 hours. As a result, the size of liposomes was 108.3 ± 5.0 nm at Lipo-DOX-Au and 94.1 ± 12.2 nm at Lipo-DOX-Au-RGD, and it was observed that doxorubicin was mounted inside the liposome up to 52%. After 6 hours of HIFU treatment, the viability of U87mg cells treated with Lipo-DOX-Au decreased by around 20% compared to Lipo-DOX, and Lipo-DOX-Au-RGD had a higher uptake rate than Lipo-DOX. In vivo study using PET images, it was confirmed that 64Cu-Lipo-DOX-Au-RGD was taken up into the tumor immediately after injection and maintained for up to 4 hours. In this study, drugs released from liposomes-gold nanoparticles via ultrasound and RGD targeting were confirmed by non-invasive imaging. In cell-level experiments, HIFU treatment of gold nanoparticle-coupled liposomes significantly decreased tumor survival, while RGD-liposomes exhibited high tumor targeting and rapid release in vivo imaging. It is expected that the combination of these models with ultrasound is served as an effective drug delivery material with therapeutic outcomes.

Dismantling and Restoration of the Celadon Stool Treasure with an Openwork Ring Design (보물 청자 투각고리문 의자의 해체 및 복원)

  • KWON, Ohyoung;LEE, Sunmyung;LEE, Jangjon;PARK, Younghwan
    • Korean Journal of Heritage: History & Science
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    • v.55 no.2
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    • pp.200-211
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    • 2022
  • The celadon stools with an openwork ring design which consist of four items as one collection were excavated from Gaeseong, Gyeonggi-do Province. The celadon stools were designated and managed as treasures due to their high arthistorical value in the form of demonstrating the excellence of celadon manufacturing techniques and the fanciful lifestyles during the Goryeo Dynasty. However, one of the items, which appeared to have been repaired and restored in the past, suffered a decline in aesthetic value due to the aging of the treatment materials and the lack of skill on the part of the conservator, raising the need for re-treatment as a result of structural instability. An examination of the conservation condition prior to conservation treatment found structural vulnerabilities because physical damage had been artificially inflicted throughout the area that was rendered defective at the time of manufacturing. The bonded surfaces for the cracked areas and detached fragments did not fit, and these areas and fragments had deteriorated because the adhesive trickled down onto the celadon surface or secondary contaminants, such as dust, were on the adhesive surface. The study identified the position, scope, and conditions of the bonded areas at the cracks UV rays and microscopy in order to investigate the condition of repair and restoration. By conducting Fourier-transform infrared spectroscopy(FT-IR) and portable x-ray fluorescence spectroscopy on the materials used for the former conservation treatment, the study confirmed the use of cellulose resins and epoxy resins as adhesives. Furthermore, the analysis revealed the addition of gypsum(CaSO4·2H2O) and bone meal(Ca10 (PO4)6(OH)2) to the adhesive to increase the bonding strength of some of the bonded areas that sustained force. Based on the results of the investigation, the conservation treatment for the artifact would focus on completely dismantling the existing bonded areas and then consolidating vulnerable areas through bonding and restoration. After removing and dismantling the prior adhesive used, the celadon stool was separated into 6 large fragments including the top and bottom, the curved legs, and some of the ring design. After dismantling, the remaining adhesive and contaminants were chemically and physically removed, and a steam cleaner was used to clean the fractured surfaces to increase the bonding efficacy of the re-bonding. The bonding of the artifact involved applying the adhesive differently depending on the bonding area and size. The cyanoacrylate resin Loctite 401 was used on the bonding area that held the positions of the fragments, while the acrylic resin Paraloid B-72 20%(in xylene) was treated on cross sections for reversibility in the areas that provided structural stability before bonding the fragments using the epoxy resin Epo-tek 301-2. For areas that would sustain force, as in the top and bottom, kaolin was added to Epo-tek 301-2 in order to reinforce the bonding strength. For the missing parts of the ring design where a continuous pattern could be assumed, a frame was made using SN-sheets, and the ring design was then modeled and restored by connecting the damaged cross section with Wood epos. Other restoration areas that occurred during bonding were treated by being filled with Wood epos for aesthetic and structural stabilization. Restored and filled areas were color-matched to avoid the feeling of disharmony from differences of texture in case of exhibitions in the future. The investigation and treatment process involving a variety of scientific technology was systematically documented so as to be utilized as basic data for the conservation and maintenance.