• Title/Summary/Keyword: 2단계 배양방법

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Indentification of Specific Proteins synthesized During Somatic Embryogenesis of Rice (벼 callus로부터 재분화 과정에서 생성되는 특수한 단백질의 동정)

  • Won, Jae Hee;Choi, Hong Jib;Kim, Dal Ung
    • Current Research on Agriculture and Life Sciences
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    • v.9
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    • pp.95-102
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    • 1991
  • This study was conducted to investigate the physiological and biochemical elements relating embryogenisis. Also we examined the differences of protein in embryogenic callus and nonembryogenic callus by SDS-PAGE and two-dimensional gel electrophoresis. In this study, we have focused attention on the process of total proteins during sometic embryogenesis of rice. When compared the proteins of embryogenic callus and nonembryogenic callus, some different proteins were observed and serval proteins were increased in embryogenic callus. Some proteins were decreased or disappeared in embryogenic callus. Near 43kD protein band was only observed in embryogenic callus. The other bands were similar to each lines. The result of two-diimensional gel electrophoresis, E-callus specific proteins were observed. This Results may indicate that these proteins were associated with somatic embryogenesis.

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Development of Chimeric Embryos Aggregated with Blastomeres from Parthenogenetic and in vitro Fertilized Bovine Embryos (소의 단위 발생란과 체외수정란 유래의 할구 응집에 의한 키메라 수정란(Chimeric Embryo)의 발달)

  • E. H. Yeao;Kim, Y. S.;Lee, S. L.;T. Y. Kang;D. O. Kwack;Lee, H. J.;S. Y. Choe
    • Journal of Embryo Transfer
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    • v.18 no.1
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    • pp.15-25
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    • 2003
  • 발생학에서 키메라(chimera)는 2개 이상의 다른 유전자형의 세포, 또는 다른 종의 세포로부터 만들어진 1개의 생물개체를 뜻하는 말로, 이는 초기 수정란의 발달과 포유류의 분화를 연구하는데 이용되고 있다. 키메라를 만드는 방법에는 할구와 내세포괴를 응집시키는 방법과 배반포 내에 여러 종류의 세포를 주입하는 방법이 있다 본 실험에서는 서로 다른 두 가지 방법의 활성화 처리법, 즉, ionomycin 처리 후 Cycloheximide (CHX) 또는 6-Dimetylaminopurine (6-DMAP)에 따른 단위 발생란의 분할과 단계적인 발달율을 살펴 보고자 하였으며, 서로 다른 방법에 의해 생산된 단위발생란 유래의 할구와 체외수정란 유래 할구를 응집하여 키메라 수정란(chimeric embryo)를 만든 후 체외수정란과 발달율을 비교해 보았다. 도축장 유래의 난소에서 난자를 채취하여 체외에서 22~24시간 성숙시킨 후 난구세포를 제거하고 metaphase II 단계의 난자를 5 $\mu$M ionomycin에 4분간 처리한 후, 10 $\mu$g/ml CHX/5 $\mu$g/ml cytochalasin B (CCB)에 5시간 또는 1.9 mM 6-DMAP에 4시간 처리하여 분할율과 배반포기 발달율을 비교 조사하였다. 난자 분할율에서는 체외수정란과 6-DMAP처리 단위 발생란에서 각각가 83.7 와 85.5%로 CHX/CCB 처리 단위발생란의 57.9%보다 유의적으로 높게(P<0.05) 나타났으며, 배반포기 발달율에 있어서는 체외수정란의 발달율이 27.8%로 6-DMAP처리 활성란 12.3%와 CHX/CCB 처리 활성란 5.3%보다 유의적으로 높게 (P<0.05) 나타났다. 키메라 수정란(chimeric embryo)은 서로 다른 두 가지 처리에 의해 생산된 단위발생란의 할구 2개와 체외수정란 유래의 할구 2개를 빈 투명대 내에서 응집시켜 제조하였다 빈 투명대 내에 키메라 수정란(chimeric embryos)의 8 세포기까지의 발달율은, 체외 수정란 할구 2개와 CHX/CCB 처리에 의한 할구 2개를 응집한 그룹은 46.1%, 체외 수정란 할구와 6-DMAP 유래 할구 2개를 응집한 그룹은 52.8% 였으며, handled control은 54.7%로 체외 수정란 77.7%에 비해 유의적으로 낮게(P<0.05) 나타났다. 배반포기까지의 발달율은 체외 수정란과 CHX/CCB에 의해 생산된 키메라 수정란(chimeric embryo)은 12.8%, 체외 수정란과 6-DMAP에 의한 키메라 수정란(chimeric embryo)은 18.8%로 handled control의 21.4%에 비해 유의적으로 낮았으며(P<0.05), 이들 키메라 수정란(chimeric embryos)은 체외 수정란의 34.9%에 비해 유의적으로 낮게(P<0.05)나타났다. 6-DMAP 처리 단위발생이 유기된 수정란 할구 2개와 체외수정란의 할구 2개의 응집에 의한 키메라 수정란(chimeric embryos)의 발달율이 CHX/CCB와 체외수정란의 응집에 의한 키메라 수정란(chimeric embryos)에 비해 다소 높게 나타났으나, 유의적인 차이는 없었다. 본 실험의 결과 서로 다른 방법에 의한 단위 발생란 유래의 할구와 체외 수정란 유래의 할구가 응집에 의한 재조합이 가능하였고 이들을 체외에서 배양하여 배반포기의 수정란까지 발달시켰다.

The Role of Interleukin 8 and NF(nuclear factor)-κB in Rhinovirus-Induced Airway Inflammation (Rhinovirus 유발성 기도염증반응에서 Interleukin-8과 전사인자 NF(nuclear factor)-κB의 역할에 대한 연구)

  • Yoon, Ho Joo;Kim, Mi Ok;Sohn, Jang Won;Kim, Jung Mogg;Shin, Dong Ho;Park, Sung Soo
    • Tuberculosis and Respiratory Diseases
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    • v.54 no.1
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    • pp.104-113
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    • 2003
  • Background : Rhinovirus(RV) infections frequently trigger dyspnea and paroxysmal cough in adult patients with asthma and are the most prevalent cause of the common cold. However, the mechanisms of a RV-induced airway inflammation is unclear. Since the RV does not directly destroy the airway epithelium, it is presumed that the immune response to the RV contributes to the pathogenesis of the respiratory symptoms. In order to test this hypothesis, this study characterized the time-sequenced alterations in interleukin(IL)-8 elaboration from the human bronchial epithelial cells and evaluated the role of NF(nuclear factor)-${\kappa}B$ in the RV-induced IL-8 production by pretreating the inhibitors of NF-${\kappa}B$ activation. Methods : The ability of RV-infected human bronchial epithelial cells and BEAS-2B cells to produce the IL-8 was compared with the controls. This study infected BEAS-2B cells with the RV14 obtained from the American Type Culture Collection. The supernatants were harvested from the RV infected BEAS-2B cells and the controls at 2hr, 4hr, 6hr, 12hr, 24hr, 48hr from the inoculation time. This study measured the IL-8 concentration using the ELISA kits. In order to elucidate the role of NF-${\kappa}B$ in the RV-induced IL-8 production, the effect of the NF-${\kappa}B$ inhibitors was evaluated on RV-induced IL-8 production. Results: The BEAS-2B cells produced small amounts of IL-8 that accumulated slowly with time in the culture. The RV was a potent stimulator of the IL-8 proteins production by BEAS-2B human bronchial epithelial cells. Antioxidants, N-acetyl-L-cysteine(NAC),\ and pyrrolidine dithiocarbamate(PDTC), blocked the IL-8 elaboration by the RV-infected BEAS-2B cells, which was dose-dependent, but N-Tosyl-L-phenylalanine chloromethyl ketone(TPCK) did not. Conclusion: Some antioxidants inhibited the RV-induced IL-8 production by blocking the NF-${\kappa}B$, which may have a therapeutic potential in asthma.

Effect of Cryopreservation of Sibling 2PN Zygotes on Cumulative Delivery Rates in the Human IVF-ET Program (전핵 시기에서의 수정란 동결이 체외수정 및 배아이식술에서의 누적 분만율에 미치는 영향)

  • Kim, Myo-Kyung;Lee, Sun-Hee;Choi, Su-Jin;Choi, Hye-Won;Park, Dong-Wook;Lim, Chun-Kyu;Song, In-Ok;Lee, Hyoung-Song
    • Clinical and Experimental Reproductive Medicine
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    • v.37 no.4
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    • pp.329-338
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    • 2010
  • Objective: This study was carried out to know whether cryopreservation of sibling 2PN zygotes could increase the cumulative delivery rates in the patients who had less than 10 fertilized zygotes. Methods: A retrospective analysis was performed in 138 in vitro fertilization-embryo transfer (IVF-ET) cycles with less than 10 fertilized zygotes during January 2003 to December 2007 in Cheil General Hospital. These cycles were divided into two groups. In Group I (n=86), all fertilized embryos were cultured to transfer on day 3 without cryopreserved embryos at the 2PN stage. In Group II (n=52), among fertilized zygotes, some sibling zygotes were frozen at the 2PN stage, the remainder were cultured to transfer. Clinical outcomes in fresh ET cycles and cumulative ongoing pregnancy rates after subsequent frozen-thawed (FT)-ET cycles were compared. Results: There were no significant differences in female mean age, number of retrieved oocytes and total fertilized embryos between two groups, Number of cultured embryos was significantly lower in Group II ($5.2{\pm}0.5$) than in Group I ($8.4{\pm}0.7$) (p<0.01). Also, number of transferred embryos was significantly lower in Group II ($3.3{\pm}0.6$) compared with Group I ($3.6{\pm}0.6$) (p<0.01). ${\beta}$-hCG positive rates and delivery rates (51.2 vs. 46.2 % and 41.9 vs. 34.6 %, respectively) after fresh ET were slightly higher in Group I than in Group II. However, the differences were not statistically significant. Also, the cumulative delivery rates after subsequent FT-ET cycles were not significantly different between Group I (48.8%) and Group II (50.0%). Conclusion: This study showed that cryopreservation of sibling 2PN zygotes from patients who had less than 10 zygotes in the fresh ET cycles did not increase cumulative delivery outcomes. But, it could provide an alternative choice for patients due to offering more chance for embryo transfers if pregnancy was failed in fresh IVF-ET cycles.

Establishment of Standard Methods for Marine Ecotoxicological Test (해양생태독성평가를 위한 표준시험방법 개발에 관한 연구)

  • Park, Gyung-Soo;Lee, Seung-Min;Han, Tae-Jun;Lee, Jung-Suk
    • The Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY
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    • v.13 no.2
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    • pp.106-111
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    • 2008
  • Six standard methods for marine ecotoxicological tests were established(or applicated) using marine decomposer, primary producers and consumers. Development processes referred to the standard methods established by USEPA(United States Environmental Protection Agency), international organizations and European methods. However, the standard test species were selected among the domestic species generally found in the Korean waters and sediments. The test methods provide the culture/maintenance of test species, test methods, reproducibility and quality acceptance criteria etc. A total of nine test species were designated including bioluminescent bacteria(Vibrio fischeri), diatom(Skeletonema costatum), seaweed(Ulva pertusa), rotifer(Brachionus plicatilis), benthic copepod(Tigriopus japonicus), benthic amphipods(Mandibulophoxus mai, Monocorophium acherusicum), and fishes(Oryzias latipes, Paralichthys olivaceus). These test species represent the decomposer, primary producer and consumers in marine trophic system in Korean coastal ecosystems, and we recommend the "battery test" including at least one species from the each trophic level for marine ecotoxicological test.

Polishing characteristics of polyetherketoneketone on Candida albicans adhesion (Polyetherketoneketone의 연마 특성이 Candida albicans의 부착에 미치는 영향)

  • Kim, Hyunyoung;Lee, Jonghyuk;Lee, Sung-Hoon;Baek, Dongheon
    • The Journal of Korean Academy of Prosthodontics
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    • v.58 no.3
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    • pp.207-216
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    • 2020
  • Purpose: To compare the polishing characteristics and their influence on Candida albicans adhesion to the recently introduced polyetherketoneketone (PEKK) and the conventional polymethylmethacrylate (PMMA) denture resin material. Materials and methods: Specimens from PEKK (Group E) and PMMA (Group M) were made in dimensions of 8 mm in diameter and 2 mm in thickness. The specimens were further divided into sub-groups according to the extent of polishing (ER, MR: rough; EP, MP: polished, N = 12 each). The specimens were polished using polishing machine and SiC foil. ER and MR group specimens were polished with 600 grit SiC foil only. EP and MP groups were further polished with 800, 1,000, 1,200 grit SiC foils sequentially. To measure the surface roughness values (Sa) of specimens, atomic force microscope (AFM) was used and scanning electron microscope (SEM) observation under 1,000, and 20,000 magnifications was performed to investigate surface topography. The polished specimens were soaked in C. albicans suspension for 2 hours with shaking to promote adhesion. The attached C. albicans were detached from the surface with 10 times of pipetting. The suspension of detached C. albicans was performed by serial dilution to 103 times, and the diluted suspensions were inoculated on Sabouraud dextrose agar plates using spread plate method. After incubating the plate for 48 hours, colony forming unit (CFU)/plate of C. albicans was counted. Statistical analysis was performed using one-way ANOVA and Tukey HSD test to confirm significant difference between the groups (α=.05). Results: Average Sa value was significantly higher in MR group compared to other groups (P<.05), meaning that additional polishing steps reduced surface roughness effectively only in the PMMA specimens. There was no significant difference in Sa values between MP and EP groups. In SEM images, PEKK specimens showed numerous spikes of abraded material protruding from the surface and this phenomenon was more significant in EP group. The mean CFU/plate value was the highest in EP group and this was significant when it was compared to MP group (P<.05) which was the lowest. Conclusion: Polishing PEKK using serial SiC abrasive foil may result in higher adhesion of C. albicans. In clinic, this should be considered carefully.

A Prospective Comparison of Fertilizability of in vitro Matured Human Oocytes Obtained from Stimulated Cycle: Conventional Versus ICSI (과배란유도 주기에서 얻어진 체외성숙 난자의 수정능: 고식적 체외수정시술과 세포질내정자주입법의 비교)

  • Jee, Byung-Chul;An, So-Jung;Moon, Jeong-Hee;Hwang, Eun-Ju;Suh, Chang-Suk;Kim, Seok-Hyun;Moon, Shin-Yong
    • Clinical and Experimental Reproductive Medicine
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    • v.36 no.4
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    • pp.249-254
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    • 2009
  • Objective: The aim of this study was to compare the fertilization and cleavage rates of human in vitro matured oocytes after fertilized by conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Methods: A total of 135 GV stage oocytes were obtained from 59 women who received ovarian stimulation and IVF during Jan 2007 to Oct 2008. Ovarian hyperstimulation was performed using hMG or recombinant FSH with GnRH antagonist and then ovulation triggered by recombinant hCG. The immature oocytes obtained from stimulation cycles were cultured in IVM medium up to 48 hrs; commercial medium supplemented with rFSH 75 mIU/mL, rhCG 0.5 IU/mL and rEGF 10 ng/mL. The in vitro matured oocytes were fertilized by conventional IVF (41 GV oocytes) or ICSI method (94 GV oocytes). Results: Maturation rate were 51.2% and 59.6% in conventional IVF group and ICSI group, respectively. There was no significant difference in fertilization rates between two groups; 71.4% and 80.4%, respectively. The cleavage rate was also similar in two groups. Conclusion: The presented data suggest that conventional IVF has comparable fertilization and cleavage potential compared with ICSI as the insemination method of immature human oocytes obtained from stimulated cycle.

Glucose 6-Phosphate Dehydrogenase Activity of Bovine Embryos Produced in vitro (소 체외수정란의 Glucose 6-Phosphate Dehydrogenase 활성)

  • Ryoo, Z.Y.;Park, H.D.;Lee, K.K.
    • Clinical and Experimental Reproductive Medicine
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    • v.22 no.3
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    • pp.301-307
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    • 1995
  • 소 체외수정란에 있어서 pentose phosphate pathway (PPP)를 연구하기 위해서, 한개의 체외수정란으로부터 glucose 6-phosphate dehydrogenase (G6PDH)의 활성을 효소증폭방법으로 측정하였다. Glucose 6-phosphate (G6P) 기질을 처리하지 않은 2, 4, 8세포기, 상실배 및 배반포기 수정란에서의 G6PDH 활성치는 각각 $25.5{\pm}3.3$, $27.8{\pm}3.4$, $40.9{\pm}6.2$, $34.9{\pm}3.6$$52.9{\pm}2.5{\times}10^{-8}mol/embryo/h$ 을 나타내었다. 즉, 8 세포기 이후 수정란들은 2 세포기나 4 세포기보다도 높은 효소활성치를 보여주었다 (P<0.01). 그리고 G6P 기질을 첨가한 2,4,8 세포기, 상실배기 및 배반포기 수정란의 G6PDH 활성치는 각각 $32.3{\pm}3.9$, $29.4{\pm}1.8$, $51.9{\pm}4.2$, $42.6{\pm}2.7$$52.9{\pm}2.5{\times}10^{-8}mol/embryo/h$ 로서 기질 무처리구와 마찬가지로 유의성이 인정되었다 (P<0.01). 전반적으로 수정란의 발달단계에 있어서 G6P 첨가한 수정란들에 G6PDH의 효소활성치가 기질을 처리하지 수정란들의 것보다도 높은 경향을 보였다. 한편, 소 체외수정란의 G6PDH 효소활성치와 발생능과의 관계를 알아보기 위하여, 4 세포기 수정란들을 효소활성치의 정량적 수준 (low, middle, high)에 따라 3 군으로 분류한 다음 $38.5^{\circ}C$, 5% $CO_2$에서 5일간 난구세포들과 공동배양을 실시하였다. 그 결과, G6PDH 효소활성치 차이에 따른 수정란들의 체외발달율에는 유의성이 인정되지 않았다. 본 실험의 결과를 종합하여 볼 때, 소 체외수정란에 있어서 PPP 대사는 8세포기 이후부터 활발히 이루어지고 있음을 알 수 있었다.

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Application of Nano Fe°-impregnated Biochar for the Stabilization of As-contaminated Soil (비소 오염토양의 안정화를 위한 나노 Fe° 담지 바이오차 적용 연구)

  • Choi, Yu-Lim;Angaru, Ganesh Kumar Reddy;Ahn, Hye-Young;Park, Kwang-Jin;Joo, Wan-Ho;Yang, Jae-Kyu;Chang, Yoon-Young
    • Journal of Environmental Impact Assessment
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    • v.29 no.5
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    • pp.350-362
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    • 2020
  • In this study, nano Fe°-impregnated biochar (INPBC) was prepared using pruning residues and one-pot synthetic method and evaluated its performance as an amendment agent for the stabilization of arsenic-contaminated soil. For the preparation of INPBC, the mixture of pruning residue and Fe (III) solution was heated to 220℃ for 3hr in a teflon-sealed autoclave followed by calcination at 600℃ under N2 atmosphere for 1hr. As-prepared INPBC was characterized using FT-IR, XRD, BET, SEM. For the stabilization test of as-prepared INPBC, As-contaminated soils (Soil-E and Soil-S) sampled from agricultural sites located respectively near E-abandoned mine and S-abandoned mine in South Korea were mixed with different of dosage of INPBC and cultivated for 4 weeks. After treatment, TCLP and SPLP tests were conducted to determine the stabilization efficiency of As in soil and showed that the stabilization efficiency was increased with increasing the INPBC dosage and the concentration of As in SPLP extractant of Soil-E was lower than the drinking water standard level of Ministry of Environment of South Korea. The sequential fractionation of As in the stabilized soils indicated that the fractions of As in the 1st and 2nd stages that correspond liable and known as bioavailable fraction were decreased and the fractions of As in 3rd and 4th stages that correspond relatively non-liable fraction were increased. Such a stabilization of As shows that the abundant nano Fe° on the surface of INPBC mixed with As-contaminated soils played the co-precipitation of As leaching from soil by surface complexation with iron. The results of this study may imply that INPBC as a promising amendments for the stabilization of As-contaminated soil play an important role.

Nuclear Remodeling and In Vitro Development of Bovine Oocytes Following Nuclear Transfer of Bovine Fetal Fibroblasts (태아 섬유아세포로 핵치환된 소 난자의 핵의 재구성과정과 체외 배발달)

  • Um, J. H.;S. J. Uhm;Kim, N-H;Lee, H. T.;K. S. Chung
    • Korean Journal of Animal Reproduction
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    • v.24 no.1
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    • pp.59-67
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    • 2000
  • This study was investigated the developmental potential of bovine embryos following nuclear transfer with bovine fetal fibroblasts (BFF). BFF were isolated from a male 45-day-old-fetus. Non-starved BFF labeled with MitoTracker were transferred into perivitelline space of enucleated oocytes. BFF-oocyte units were fused by electric pulse, and then fused oocytes were activated with calcium ionophore A23187 and subsequently 6-dimethylaminopurine (6-DMAP). The resulting zygotes were placed into CRlaa bovine embryo culture medium. Transfer of the nucleus into enucleated oocyte led to premature chromosome condensation, swelling and pronucleus formation. Remodeled oocytes were developed to the mitotic and 2-cell stage at 18 to 26 h after nuclear transfer. The incidence of in vitro development to the blastocyst stages was 21% of fused oocytes. Mitochondria of BFF eliminated rapidly and were not detected at 8 h after fusion. These results suggest that BFF can be successfully reprogrammed in enucleated bovine oocytes, and that reconstructed embryos can develop to the blastocyst stage.

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