• Title/Summary/Keyword: Single Cell

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Production of homozygous klotho knockout porcine embryos cloned from genome-edited porcine fibroblasts

  • Lee, Sanghoon;Jung, Min Hee;Oh, Hyun Ju;Koo, Ok Jae;Park, Se Chang;Lee, Byeong Chun
    • Journal of Embryo Transfer
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    • v.31 no.3
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    • pp.179-183
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    • 2016
  • Even though klotho deficiency in mice exhibits multiple aging-like phenotypes, studies using large animal models such as pigs, which have many similarities to humans, have been limited due to the absence of cell lines or animal models. The objective of this study was to generate homozygous klotho knockout porcine cell lines and cloned embryos. A CRISPR sgRNA specific for the klotho gene was designed and sgRNA (targeting exon 3 of klotho) and Cas9 RNPs were transfected into porcine fibroblasts. The transfected fibroblasts were then used for single cell colony formation and 9 single cell-derived colonies were established. In a T7 endonuclease I mutation assay, 5 colonies (#3, #4, #5, #7 and #9) were confirmed as mutated. These 5 colonies were subsequently analyzed by deep sequencing for determination of homozygous mutated colonies and 4 (#3, #4, #5 and #9) from 5 colonies contained homozygous modifications. Somatic cell nuclear transfer was performed to generate homozygous klotho knockout cloned embryos by using one homozygous mutation colony (#9); the cleavage and blastocyst formation rates were 72.0% and 8.3%, respectively. Two cloned embryos derived from a homozygous klotho knockout cell line (#9) were subjected to deep sequencing and they showed the same mutation pattern as the donor cell line. In conclusion, we produced homozygous klotho knockout porcine embryos cloned from genome-edited porcine fibroblasts.

Interleukin-10 Polymorphisms in Association with Prognosis in Patients with B-Cell Lymphoma Treated by R-CHOP

  • Kim, Min Kyeong;Yoo, Kyong-Ah;Park, Eun Young;Joo, Jungnam;Lee, Eun Young;Eom, Hyeon-Seok;Kong, Sun-Young
    • Genomics & Informatics
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    • v.14 no.4
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    • pp.205-210
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    • 2016
  • Interleukin-10 (IL10) plays an important role in initiating and maintaining an appropriate immune response to non-Hodgkin lymphoma (NHL). Previous studies have revealed that the transcription of IL10 mRNA and its protein expression may be infl uenced by several single-nucleotide polymorphisms in the promoter and intron regions, including rs1800896, rs1800871, and rs1800872. However, the impact of polymorphisms of the IL10 gene on NHL prognosis has not been fully elucidated. Here, we investigated the association between IL10 polymorphisms and NHL prognosis. This study involved 112 NHL patients treated at the National Cancer Center, Korea. The median age was 57 years, and 70 patients (62.5%) were men. Clinical characteristics, including age, performance status, stage, and extra-nodal involvement, as well as cell lineage and International Prognostic Index (IPI), were evaluated. A total of four polymorphisms in IL10 with heterozygous alleles were analyzed for hazard ratios of overall survival (OS) and progression-free survival (PFS) using Cox proportional hazards regression analysis. Diffuse large B-cell lymphoma was the most common histologic type (n = 83), followed by T-cell lymphoma (n = 18), mantle cell lymphoma (n = 6), and others (n = 5). Cell lineage, IPI, and extra-nodal involvement were predictors of prognosis. In the additive genetic model results for each IL10 polymorphism, the rs1800871 and rs1800872 polymorphisms represented a marginal association with OS (p = 0.09 and p = 0.06) and PFS (p = 0.05 and p = 0.08) in B-cell lymphoma patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). These findings suggest that IL10 polymorphisms might be prognostic indicators for patients with B-cell NHL treated with R-CHOP.

The Effects of ASMase Mediated Endothelial Cell Apoptosis in Multiple Hypofractionated Irradiations in CT26 Tumor Bearing Mice

  • Zhu, Hong;Deng, Kai;Zhao, Ya-Qin;Wang, Xin;Shen, Ya-Li;Liu, Tai-Guo;Cui, Dan-Dan;Xu, Feng
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.11
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    • pp.4543-4548
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    • 2015
  • Background: To investigate the effects of ASMase mediated endothelial cell apoptosis in multiple hypofractionated irradiations in CT26 tumor bearing mice. Materials and Methods: Thirty-five CT26 tumor bearing mice were subjected to single ionizing radiation (IR) of 0, 3, 6, 9, 12, 15, 18 Gy. Eight hours after IR, the mice were sacrificed and tumor tissues were used for CD31 immunohistochemistry staining, TUNEL and CD31 double staining, ASMase activity assay. Then 6 and 12 Gy were chosen for multiple hypofractionated IR experiments according to the above results. Each time after IR, 5 mice were sacrificed and assayed as above. Results: The ASMase activities were increased significantly after a single IR of 12 Gy or higher which was accompanied with remarkable increased endothelial cell apoptosis and decreased MVD. For 6 Gy which was not high enough to trigger ASMase activation, after 2 or more times of IR, the ASMase activities were significantly increased accompanied with remarkable increased endothelial cell apoptosis and decreased MVD. While for 12 Gy, after 2 or more times of IR, the ASMase activities and endothelial cell apoptosis rates were maintained without remarkable increase; however, the MVD was significantly decreased. What's more, the cancer cell apoptosis rates were significantly increased after multiple IR for both 6 Gy and 12 Gy. Conclusions: ASMase mediated endothelial cell apoptosis may play an important role in the process of multiple hypofractionated IR for CT26 colorectal carcinoma.

Biocompatible Formation of Silica/Titania Nanocomposite Shells on Living Chlorella Cells

  • Go, Eun-Hye;Yun, Yeon-Jeong;Jin, Seung-Uk;Hwang, Ji-Min;Lee, Gyu-Nam;Yang, Seong-Ho;Choe, In-Seong
    • Proceedings of the Korean Vacuum Society Conference
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    • 2012.02a
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    • pp.553-553
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    • 2012
  • The artificial shells of hard inorganic nanocomposites on individual cells would protect the cells physically and chemically, and control cell division. These emerging properties could be combined with cell-surface functionalizations for applications to cell-based sensors and assays as well as for fundamental studies on single-cell biology. In this work, individual Chlorella cells were encapsulated within a silica/titania nanocomposite shell in a biocompatible fashion that utilized a designed peptide, RKKRKKRKKRKKDDDDDDDD, as a catalytic template for formation of both $SiO_2$ and $TiO_2$ on the cell surface. The cell viability was maintained, and the division of the encapsulated Chlorella cells was controlled. The cell viability was enhanced compared with the $TiO_2$-shell formation. In addition, the incorporation of $TiO_2$ to the shell made it possible to anchor the ligands of interest to the shell via catechol chemistry. All in all, the combination of biological $SiO_2$ and abiolgical $TiO_2$ for the shell formation gave more tunability of the artificial shells compared with the $SiO_2$ or $TiO_2$ shells only.

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Enhancement of aberrantly modified integrin-mediated cell motility in multicellular tumor spheroids

  • BYOUNGJAE KIM;NU-RI IM;TAESEOK DANIEL YANG;JIAN KIM;KWANG-YOON JUNG;TAE HOON KIM;SEUNG-KUK BAEK
    • International Journal of Oncology
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    • v.56 no.6
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    • pp.1490-1498
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    • 2020
  • Multicellular tumor spheroids (MTSs) of malignant cells can display cell-cell and cell-matrix interactions, different from monolayer cultures. The objective of the present study was to examine difference in intercellular and cell-matrix interaction between monolayered cultures and spheroid cultures. Expression levels of cell adhesion molecules (CAMs) and epithelial-mesenchymal transition (EMT) signaling molecules in monolayered cells and MTS cells were compared. The motility of single cells dispersed from each culture was evaluated using a live-cell imaging device. The effect of an E-cadherin neutralizing antibody, DECMA, was also compared between the two cultures. Among various CAMs, only E-cadherin was increased in MTSs. The motility of single cells dispersed from MTSs was higher than that from monolayered cells. Compared with monolayered cells, the molecular weight (MW) of β1 integrin was decreased during MTS formation, particularly during the early stage. This notable reduction was maintained when DECMA was used to treat MTSs. Additionally, the expression levels of the EMT signaling molecules Snail and ILK increased more in MTSs than in monolayered cells. The blocking of E-cadherin elicited increased expression levels of EMT molecules and cellular motility only in MTSs. In conclusion, the alteration of E-cadherin expression and presence of low-MW β1 integrin in MTS may enhance cell motility via the upregulation of EMT signaling molecules that may be intensified by blocking E-cadherin.

Modeling Growth Kinetics of Lactic Acid Bacteria for Food Fermentation

  • Chung, Dong-Hwa;Kim, Myoung-Dong;Kim, Dae-Ok;Koh, Young-Ho;Seo, Jin-Ho
    • Food Science and Biotechnology
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    • v.15 no.5
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    • pp.664-671
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    • 2006
  • Modeling the growth kinetics of lactic acid bacteria (LAB), one of the most valuable microbial groups in the food industry, has been actively pursued in order to understand, control, and optimize the relevant fermentation processes. Most modeling approaches have focused on the development of single population models. Primary single population models provide fundamental kinetic information on the proliferation of a primary LAB species, the effects of biological factors on cell inhibition, and the metabolic reactions associated with cell growth. Secondary single population models can evaluate the dependence of primary model parameters, such as the maximum specific growth rate of LAB, on the initial external environmental conditions. This review elucidates some of the most important single population models that are conveniently applicable to the LAB fermentation analyses. Also, a well-defined mixed population model is presented as a valuable tool for assessing potential microbial interactions during fermentation with multiple LAB species.

A Study on Inset Fed Microstrip Antenna Loaded with Complementary Single Loop Resonator (CSLR을 갖는 인셋 급전 마이크로스트립 안테나에 관한 연구)

  • Hong, Jae-Pyo;Kim, Byung-Mun
    • The Journal of the Korea institute of electronic communication sciences
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    • v.9 no.8
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    • pp.921-926
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    • 2014
  • In this paper, the characteristics of inset fed microstrip antenna loaded with CSLR(complementary single loop resonator) are studied. Effective permeability parameters of the SLR unit cell is retrieved from simulated scattering parameters, and structure parameters of the SLR unit cell are selected so that effective permeability is negative value at the operating frequency. The optimized inset fed microstrip antenna loaded with SLR for a $3{\times}3$ array in the ground plane of a conventional patch antenna is designed and simulation results of return loss and radiation pattern are shown. At resonant frequency 2.82 GHz, the overall dimension of the proposed antenna is reduced by approximately 56.8% compared to the conventional inset fed antenna. Simulation results are obtained by 3D FEM solver(Ansoft's HFSS).