• Title/Summary/Keyword: Label-Free

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Development of a Label-Free LC-MS/MS-Based Glucosylceramide Synthase Assay and Its Application to Inhibitors Screening for Ceramide-Related Diseases

  • Fu, Zhicheng;Yun, So Yoon;Won, Jong Hoon;Back, Moon Jung;Jang, Ji Min;Ha, Hae Chan;Lee, Hae Kyung;Shin, In Chul;Kim, Ju Yeun;Kim, Hee Soo;Kim, Dae Kyong
    • Biomolecules & Therapeutics
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    • v.27 no.2
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    • pp.193-200
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    • 2019
  • Ceramide metabolism is known to be an essential etiology for various diseases, such as atopic dermatitis and Gaucher disease. Glucosylceramide synthase (GCS) is a key enzyme for the synthesis of glucosylceramide (GlcCer), which is a main ceramide metabolism pathway in mammalian cells. In this article, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine GCS activity using synthetic non-natural sphingolipid C8-ceramide as a substrate. The reaction products, C8-GlcCer for GCS, could be separated on a C18 column by reverse-phase high-performance liquid chromatography (HPLC). Quantification was conducted using the multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z $588.6{\rightarrow}264.4$ for C8-GlcCer at positive ionization mode. The calibration curve was established over the range of 0.625-160 ng/mL, and the correlation coefficient was larger than 0.999. This method was successfully applied to detect GCS in the human hepatocellular carcinoma cell line (HepG2 cells) and mouse peripheral blood mononuclear cells. We also evaluated the inhibition degree of a known GCS inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on GCS enzymatic activity and proved that this method could be successfully applied to GCS inhibitor screening of preventive and therapeutic drugs for ceramide metabolism diseases, such as atopic dermatitis and Gaucher disease.

Proposal of autonomous take-off drone algorithm using deep learning (딥러닝을 이용한 자율 이륙 드론 알고리즘 제안)

  • Lee, Jong-Gu;Jang, Min-Seok;Lee, Yon-Sik
    • Journal of the Korea Institute of Information and Communication Engineering
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    • v.25 no.2
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    • pp.187-192
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    • 2021
  • This study proposes a system for take-off in a forest or similar complex environment using an object detector. In the simulator, a raspberry pi is mounted on a quadcopter with a length of 550mm between motors on a diagonal line, and the experiment is conducted based on edge computing. As for the images to be used for learning, about 150 images of 640⁎480 size were obtained by selecting three points inside Kunsan University, and then converting them to black and white, and pre-processing the binarization by placing a boundary value of 127. After that, we trained the SSD_Inception model. In the simulation, as a result of the experiment of taking off the drone through the model trained with the verification image as an input, a trajectory similar to the takeoff was drawn using the label.

LC-MS/MS-based Proteomic Analysis of Locally Advanced Rectal Tumors to Identify Biomarkers for Predicting Tumor Response to Neoadjuvant Chemoradiotherapy

  • Kim, Kyung-Ok;Duong, Van-An;Han, Na-Young;Park, Jong-Moon;Kim, Jung Ho;Lee, Hookeun;Baek, Jeong-Heum
    • Mass Spectrometry Letters
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    • v.13 no.3
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    • pp.84-94
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    • 2022
  • Neoadjuvant chemoradiotherapy (nCRT) is a standard therapy used for locally advanced rectal cancer prior to surgery, which can more effectively reduce the locoregional recurrence rate and radiation toxicity compared to postoperative chemoradiotherapy. The response of patients to nCRT varies, and thus, robust biomarkers for predicting a pathological complete response are necessary. This study aimed to identify possible biomarkers involved in the complete response/non-response of rectal cancer patients to nCRT. Comparative proteomic analysis was performed on rectal tissue samples before and after nCRT. Proteins were extracted for label-free proteomic analysis. Western blot and real-time PCR were performed using rectal cancer cell line SNU-503 and radiation-resistant rectal cancer cell line SNU-503R80Gy. A total of 135 up- and 93 down-regulated proteins were identified in the complete response group. Six possible biomarkers were selected to evaluate the expression of proteins and mRNA in SNU-503 and SNU-503R80Gy cell lines. Lyso-phosphatidylcholine acyltransferase 2, annexin A13, aldo-ketose reductase family 1 member B1, and cathelicidin antimicrobial peptide appeared to be potential biomarkers for predicting a pathological complete response to nCRT. This study identified differentially expressed proteins and some potential biomarkers in the complete response group, which would be further validated in future studies.

Algebraic Formal Specification and Formal Validation of the Standard and an Implementation of the OSPF Protocol (OSPF Protocol 표준 및 구현의 대수 정형적 명세 및 정형적 검증)

  • 박재현
    • The Journal of Korean Institute of Communications and Information Sciences
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    • v.29 no.3B
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    • pp.360-374
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    • 2004
  • The OSPF protocol is the most widely used Interior Gateway Routing Protocol. Therefore, for the reliability of behavior of gigabit swiching routers, it is essential to guarantee the interoperability and the safety of the OSPF protocol. In this paper, we analyze the standard document of the OSPF protocol, so that we provide a formal specification that specifies the protocol behaviors by detailed design level using the algebraic formal method. By referring available source codes of the OSPF protocol, we supplement the formal specification to express more detailed behaviors that is not specified definitely in the standard. We also formally verify the interoperability and the safety of the protocol state machine of the specification. By showing that the formal specification specify all of the states and the transition events that appear in the standard document of the OSPF protocol, we prove that the state machine has the completeness, and prove it has the interoperability. To prove that the specification of the protocol has the safety, we formally verify the reachability, the liveness, the livelock-free property, and the deadlock-free property. As a result, we prove the protocol has the consistency. The specification and the validation are also effective to the OSPF Version 3 that inherit the protocol mechanism of the OSPF Version 2.

N-glycoproteomic analysis of human follicular fluid during natural and stimulated cycles in patients undergoing in vitro fertilization

  • Lim, Hee-Joung;Seok, Ae Eun;Han, Jiyou;Lee, Jiyeong;Lee, Sungeun;Kang, Hee-Gyoo;Cha, Byung Heun;Yang, Yunseok
    • Clinical and Experimental Reproductive Medicine
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    • v.44 no.2
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    • pp.63-72
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    • 2017
  • Objective: Hyperstimulation methods are broadly used for in vitro fertilization (IVF) in patients with infertility; however, the side effects associated with these therapies, such as ovarian hyperstimulation syndrome (OHSS), have not been well studied. N-glycoproteomes are subproteomes used for the remote sensing of ovarian stimulation in follicular growth. Glycoproteomic variation in human follicular fluid (hFF) has not been evaluated. In this study, we aimed to identify and quantify the glycoproteomes and N-glycoproteins (N-GPs) in natural and stimulated hFF using label-free nano-liquid chromatography/electrospray ionization-quad time-of-flight mass spectrometry. Methods: For profiling of the total proteome and glycoproteome, pooled protein samples from natural and stimulated hFF samples were selectively isolated using hydrazide chemistry to obtain the total proteomes and glycoproteomes. N-GPs were validated by the consensus sequence N-X-S/T (92.2% specificity for the N-glycomotif at p<0.05). All data were compared between natural versus hyperstimulated hFF samples. Results: We detected 41 and 44 N-GPs in the natural and stimulated hFF samples, respectively. Importantly, we identified 11 N-GPs with greater than two-fold upregulation in stimulated hFF samples compared to natural hFF samples. We also validated the novel N-GPs thyroxine-binding globulin, vitamin D-binding protein, and complement proteins C3 and C9. Conclusion: We identified and classified N-GPs in hFF to improve our understanding of follicular physiology in patients requiring assisted reproduction. Our results provided important insights into the prevention of hyperstimulation side effects, such as OHSS.

Metal Oxide Thin Film Transistor with Porous Silver Nanowire Top Gate Electrode for Label-Free Bio-Relevant Molecules Detection

  • Yu, Tae-Hui;Kim, Jeong-Hyeok;Sang, Byeong-In;Choe, Won-Guk;Hwang, Do-Gyeong
    • Proceedings of the Korean Vacuum Society Conference
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    • 2016.02a
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    • pp.268-268
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    • 2016
  • Chemical sensors have attracted much attention due to their various applications such as agriculture product, cosmetic and pharmaceutical components and clinical control. A conventional chemical and biological sensor is consists of fluorescent dye, optical light sources, and photodetector to quantify the extent of concentration. Such complicated system leads to rising cost and slow response time. Until now, the most contemporary thin film transistors (TFTs) are used in the field of flat panel display technology for switching device. Some papers have reported that an interesting alternative to flat panel display technology is chemical sensor technology. Recent advances in chemical detection study for using TFTs, benefits from overwhelming progress made in organic thin film transistors (OTFTs) electronic, have been studied alternative to current optical detection system. However numerous problems still remain especially the long-term stability and lack of reliability. On the other hand, the utilization of metal oxide transistor technology in chemical sensors is substantially promising owing to many advantages such as outstanding electrical performance, flexible device, and transparency. The top-gate structure transistor indicated long-term atmosphere stability and reliability because insulator layer is deposited on the top of semiconductor layer, as an effective mechanical and chemical protection. We report on the fabrication of InGaZnO TFTs with silver nanowire as the top gate electrode for the aim of chemical materials detection by monitoring change of electrical properties. We demonstrated that the improved sensitivity characteristics are related to the employment of a unique combination of nano materials. The silver nanowire top-gate InGaZnO TFTs used in this study features the following advantages: i) high sensitivity, ii) long-term stability in atmosphere and buffer solution iii) no necessary additional electrode and iv) simple fabrication process by spray.

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A Randomized Controlled Trial Comparing Clinical Outcomes and Toxicity of Lobaplatin- Versus Cisplatin-Based Concurrent Chemotherapy Plus Radiotherapy and High-Dose-Rate Brachytherapy for FIGO Stage II and III Cervical Cancer

  • Wang, Ji-Quan;Wang, Tao;Shi, Fan;Yang, Yun-Yi;Su, Jin;Chai, Yan-Lan;Liu, Zi
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.14
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    • pp.5957-5961
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    • 2015
  • Background: We designed this randomized controlled trial (RCT) to assess whether lobaplatin-based concurrent chemotherapy might be superior to cisplatin-based concurrent chemotherapy for FIGO stage II and III cervical cancer in terms of efficacy and safety. Materials and Methods: This prospective, open-label RCT aims to enroll 180 patients with FIGO stage II and III cervical cancer, randomly allocated to one of the three treatment groups (cisplatin $15mg/m^2$, cisplatin $20mg/m^2$ and lobaplatin $35mg/m^2$), with 60 patients in each group. All patients will receive external beam irradiation (EBRT) and high-dose-rate intracavitary brachytherapy (HDR-ICBT). Patients in cisplatin $15mg/m^2$ and $20mg/m^2$ groups will be administered four cycles of $15mg/m^2$ or $20mg/m^2$ cisplatin intravenously once weekly from the second week to the fifth week during EBRT, while patients inthe lobaplatin $35mg/m^2$ group will be administered two cycles of $35mg/m^2$ lobaplatin intravenously in the second and fifth week respectively during pelvic EBRT. All participants will be followed up for at least 12 months. Complete remission rate and progression-free survival (PFS) will be the primary endpoints. Overall survival (OS), incidence of adverse events (AEs), and quality of life will be the secondary endpoints. Results: Between March 2013 and March 2014, a total of 61 patients with FIGO stage II and III cervical cancer were randomly assigned to cisplatin $15mg/m^2$ group (n=21), cisplatin $20mg/m^2$ group (n=21) and lobaplatin $35mg/m^2$ group (n=19). We conducted a preliminary analysis of the results. Similar rates of complete remission and grades 3-4 gastrointestinal reactions were observed for the three treatment groups (P=0.801 and 0.793, respectively). Grade 3-4 hematologic toxicity was more frequent in the lobaplatin group than the cisplatin group. Conclusions: This proposed study will be the first RCT to evaluate whether lobaplatin-based chemoraiotherapy will have beneficial effects, compared with cisplatin-based chemoradiotherapy, on complete remission rate, PFS, OS, AEs and quality of life for FIGO stage II and III cervical cancer.

Inflammation Scan Using $^{99m}Tc-HMPAO$ Labelled Leukocytes ($^{99m}Tc-HMPAO$를 이용한 자가백혈구표지 및 그를 이용한 염증병소의 스캔)

  • Yang, Woo-Jin;Chung, Soo-Kyo;Shinn, Kyung-Sub;Bahk, Yong-Whee;Kim, Hoon-Kyo
    • The Korean Journal of Nuclear Medicine
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    • v.23 no.2
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    • pp.219-223
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    • 1989
  • Inflammation scan using radiolabelled leukocytes has high sensitivity and specificity. Several methods for labelling leukocytes have been evaluated using P-32 diisopropyl fluorophosphate (DFP-32), H-3 thymidine, Cr-51 chromate, Ga-67 citrate and Tc-99m-sulfur colloid. In-111-oxine has proved so far to be the most reliable agent for labelling leukocytes. In-111-oxine is, however, expensive, not easily available when needed, and its radiation dose to leukocytes is relatively high. Moreover, resolution of the resultant image is relatively poor. Tc-99m is still the agent of choice because of, as compared with the indium, its favorable physical characteristics, lower cost and availability. Now the technique for labelling the leukocytes with technetium is successfully obtained using the lipophilic HAPAO with higher efficiency for granulocytes than for other cells. With this technique it is possible to label leukocytes in plasma to improve the viability of the leukocytes. Inflammation scan using Tc-99m-HMPAO has been evaluated in several laboratories, and difference in methods for separation and labelling accounts for difference in efficiency, viability and biodistribution of the labelled leukocytes. We performed inflammation scan using leukocytes labelled with Tc-99m-HMPAO in three dogs 24 hours after inoculation of live E. Coli and A. Aureus in their right abdominal wall. We separated mixed leukocytes by simple sedimentation using 6% hetastarch (HES) and labelled the leukocytes with Tc-99m-HMPAO in 20% cell free plama diluted with phosphate buffer solution(Fig. 1). Uptake was high in the liver and spleen but is was minimal in the lungs on whole body scan. Kidneys and intestine showed minimal activity although it was high in the urinary bladder(Fig. 2). Uptake of labelled leukocytes in the inflammation site was do(mite on 2 hour-postinjection scan and abscess was clearly delineated on 24 hour-delayed scan with high target-to-nontarget ratio(Fig. 3, 4). Inflammation scan using mixed leukocytes labelled with Tc-99m-HMPAO is very sensitive and specific in early detection of inflammation.

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Biosensor System for the Detection of Agrichemicals and Its Applications (농약 검출을 위한 바이오센서 시스템 연구 및 그 응용)

  • Park, Tae-Jung;Yang, Min-Ho;Lee, Sang-Yup;Kim, Soo-Hyun
    • KSBB Journal
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    • v.24 no.3
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    • pp.227-238
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    • 2009
  • In the recent years, some organic toxic chemicals were used for obtaining high-yield productivity in agriculture. The undegraded pesticides may remain in the agricultural foods through atmosphere, water, and soil and cause public health problems to environmental resources and human beings even at very low concentrations. Small amounts of pesticides can affect a central nervous system, resulting in immunogenic diseases, infertility problems, respiratory diseases and born marrow diseases, which can lead even to death. Monitoring of the environmental pesticide is one of the important issues for the human well-being. Several kinds of biosensors have been successfully applied to the detection of agrichemical toxicity. Also, few platforms for biocide detection have been definitely developed for the degradation and reaction of pesticides. Biochip and electrochemistry experiments involve immobilizing a receptor molecule on a solid substrate surface, and monitoring its interaction with an analyze in a sample solution. Furthermore, nanotechnology can be applied to make high-throughput analyses that are smaller, faster and sensitive than conventional assays. Some nanomaterials or nanofabricated surfaces can be coupled to biomolecules and used in antibody-based assays and enzymatic methods for pesticide residues. The operation procedure has become more convenient as it does not require labeling procedure. In this paper, we review the recent advances in agrichemical defection research and also describe the label-free biosensor for pesticides using various useful detection methods.

Detection of Undeclared Betamethasone Derivatives in Cosmetic Products Labeled to Contain Zinc Pyrithione as the Active Ingredient (아연피리치온을 유효성분으로 표기한 화장품류에서 미표기 성분인 베타메타손 유도체의 검출)

  • Lee, Jeong-Pyo;Park, Sung-Hwan;Yang, Seong-Jun;Kim, Sun-Mi;Son, Kyung-Hun;Yun, Mi-Ok;Choi, Sang-Sook
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.35 no.1
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    • pp.11-17
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    • 2009
  • Betamethasone propionate, an anti-inflammatory glucocorticosteroid, was detected in cosmetics with no indication on the label of this compound as an ingredient. The product was formulated as a topical spray or shampoo and labeled to contain zinc pyrithione as the active ingredient. A thin-layer chromatographic analysis was carried out on silica gel plates to provide a first indication about the presence of a compound with steroid structure and reactivity; then high-performance liquid chromatography (HPLC) separation allowed the identification of the corticosteroid agent and its quantification. To identify the corticosteroid agent from these commercial samples we collected the fractions suspected to have ketol steroids by prep HPLC and identified the compound as betamethasone propionate by NMR and MS spectrometry. Then we synthesized the standard for the betamethasone 17-propionate and 21-propionate and quantitate the corticosteroids from the sample by HPLC with that standards. By this method we identified the corticosteroid compounds from some commercial cosmetics such as zinc pyrithione sprays. The finding of betamethasone propionate in the products was shown by comparison to an authenticated standard of betamethasone propionate by retention time on reverse-phase HPLC. Two of the tested products contained betamethasone propionate at the levels of 0.005 ${\sim}$ 0.02% and the others were free of betamethasone propionate.