• Title/Summary/Keyword: Single cell analysis/methods

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Lung Adenocarcinoma Gene Mutation in Koreans: Detection Using Next Generation Sequence Analysis Technique and Analysis of Concordance with Existing Genetic Test Methods (한국인의 폐선암 유전자 돌연변이: 차세대 염기서열 분석법을 이용한 검출 및 기존 유전자 검사법과의 일치도 분석)

  • Jae Ha BAEK;Kyu Bong CHO
    • Korean Journal of Clinical Laboratory Science
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    • v.55 no.1
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    • pp.16-28
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    • 2023
  • Lung adenocarcinoma accounts for about 40% of all lung cancers. With the recent development of gene profiling technology, studies on mutations in oncogenes and tumor suppressor genes, which are important for the development and growth of tumors, have been actively conducted. Companion diagnosis using next-generation sequencing helps improve survival with targeted therapy. In this study, formalin-fixed paraffin-embedded tissues of non-small cell lung cancer patients were subjected to hematoxylin and eosin staining for detecting genetic mutations that induce lung adenocarcinoma in Koreans. Immunohistochemical staining was also performed to accurately classify lung adenocarcinoma tissues. Based on the results, next-generation sequencing was applied to analyze the types and patterns of genetic mutations, and the association with smoking was established as the most representative cause of lung cancer. Results of next-generation sequencing analysis confirmed the single nucleotide variations, copy number variations, and gene rearrangements. In order to validate the reliability of next-generation sequencing, we additionally performed the existing genetic testing methods (polymerase chain reaction-epidermal growth factor receptor, immunohistochemistry-anaplastic lymphoma kinase (D5F3), and fluorescence in situ hybridiation-receptor tyrosine kinase 1 tests) to confirm the concordance rates with the next-generation sequencing test results. This study demonstrates that next-generation sequencing of lung adenocarcinoma patients simultaneously identifies mutation.

Selection of Tropane Alkaloids High-Producing Lines by Single Cell Cloning of Hyoscyamus niger L. Root Cultures

  • Min, Ji-Yun;Park, Dong-Jin;Jeong, Mi-Jin;Song, Hyun-Jin;Kang, Seung-Mi;Kang, Young-Min;Choi, Myung-Suk
    • Journal of Korean Society of Forest Science
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    • v.98 no.2
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    • pp.142-147
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    • 2009
  • Hyoscyamus species is sources of the hypnotic and sedative drugs hyoscyamine and scopolamine. Single cells of Hyoscyamus niger were dissociated from suspension cultures and adventitious roots obtained from single-cell clones which were cultured on B5 medium containing 3% (w/v) sucrose, 0.1 mg/L IBA and 0.4% (w/v) gelrite. H. niger adventitious root lines showed wide variation in tropane alkaloids production and growth. An effective selection of 200 root lines was made possible by the application of the 'Dragendorff's reagent' for qualitative detection of the alkaloids from root. A high correlation coefficient (r=0.9390) was observed between the values obtained with the two methods based on HPLC and Dragendorff's reagent analysis. Among the selected roots, the highest scopolamine content was 16.64 mg/g DW (Hn-59), which was 8.82-fold more productive than the lowest alkaloid producing line (Hn-25). Here, we established an efficient selection method on tropane alkaloids production and suggest that the Dragendorff's reagent is of great practical value in selection of invisible compounds.

Identification of Single Nucleotide Polymorphism Marker and Association Analysis of Marbling Score in Fas Gene of Hanwoo

  • Kim, Seung-Chang;Lee, Seung-Hwan;Lee, Ji-Woong;Kim, Tae-Hun;Choi, Bong-Hwan
    • Asian-Australasian Journal of Animal Sciences
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    • v.29 no.1
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    • pp.23-28
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    • 2016
  • The Fas (APO-1, TNFRSF6) gene known as a member of the tumor necrosis factor receptor superfamily was selected for DNA marker development in Korean cattle. It is a cell membrane protein and mediates programmed cell death (apoptosis). We discovered single nucleotide polymorphisms (SNPs) within Fas gene in order to develop novel DNA markers related to economical traits at the genomic level. The sequences of whole exon and 1 kb range of both front and back of the gene were determined by direct-sequencing methods using 24 cattle. A total of 55 SNPs were discovered and we selected 31 common polymorphic sites considering their allele frequencies, haplotype-tagging status and linkage disequilibrium (LD) for genotyping in larger-scale subjects. The SNPs were confirmed genotype through the SNaPshot method (n = 274) and were examined for a possible genetic association between Fas polymorphisms and marbling score. So, the SNPs that were identified significant are g.30256G>C, g.31474C>A, g.31940A>G, and g.32982G>A. These results suggest that SNPs of Fas gene were associated with intramuscular fat content of meat quality traits in Korean cattle.

Mutation Screening and Association Study of the Folylpolyglutamate Synthetase (FPGS) Gene with Susceptibility to Childhood Acute Lymphoblastic Leukemia

  • Piwkham, Duangjai;Siriboonpiputtana, Teerapong;Beuten, Joke;Pakakasama, Samart;Gelfond, Jonathan AL;Paisooksantivatana, Karan;Tomlinson, Gail E;Rerkamnuaychoke, Budsaba
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.11
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    • pp.4727-4732
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    • 2015
  • Background: Folylpolyglutamate synthetase (FPGS), an important enzyme in the folate metabolic pathway, plays a central role in intracellular accumulation of folate and antifolate in several mammalian cell types. Loss of FPGS activity results in decreased cellular levels of antifolates and consequently to polyglutamatable antifolates in acute lymphoblastic leukemia (ALL). Materials and Methods: During May 1997 and December 2003, 134 children diagnosed with ALL were recruited from one hospital in Thailand. We performed a mutation analysis in the coding regions of the FPGS gene and the association between single nucleotide polymorphisms (SNPs) within FPGS in a case-control sample of childhood ALL patients. Mutation screening was conducted by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and subsequently with direct sequencing (n=72). Association analysis between common FPGS variants and ALL risk was done in 98 childhood ALL cases and 95 healthy volunteers recruited as controls. Results: Seven SNPs in the FPGS coding region were identified by mutation analysis, 3 of which (IVS13+55C>T, g.1297T>G, and g.1508C>T) were recognized as novel SNPs. Association analysis revealed 3 of 6 SNPs to confer significant increase in ALL risk these being rs7039798 (p=0.014, OR=2.14), rs1544105 (p=0.010, OR= 2.24), and rs10106 (p=0.026, OR=1.99). Conclusions: These findings suggested that common genetic polymorphisms in the FPGS coding region including rs7039789, rs1544105, and rs10106 are significantly associated with increased ALL risk in Thai children.

Non-invasive evaluation of embryo quality for the selection of transferable embryos in human in vitro fertilization-embryo transfer

  • Jihyun Kim;Jaewang Lee;Jin Hyun Jun
    • Clinical and Experimental Reproductive Medicine
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    • v.49 no.4
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    • pp.225-238
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    • 2022
  • The ultimate goal of human assisted reproductive technology is to achieve a healthy pregnancy and birth, ideally from the selection and transfer of a single competent embryo. Recently, techniques for efficiently evaluating the state and quality of preimplantation embryos using time-lapse imaging systems have been applied. Artificial intelligence programs based on deep learning technology and big data analysis of time-lapse monitoring system during in vitro culture of preimplantation embryos have also been rapidly developed. In addition, several molecular markers of the secretome have been successfully analyzed in spent embryo culture media, which could easily be obtained during in vitro embryo culture. It is also possible to analyze small amounts of cell-free nucleic acids, mitochondrial nucleic acids, miRNA, and long non-coding RNA derived from embryos using real-time polymerase chain reaction (PCR) or digital PCR, as well as next-generation sequencing. Various efforts are being made to use non-invasive evaluation of embryo quality (NiEEQ) to select the embryo with the best developmental competence. However, each NiEEQ method has some limitations that should be evaluated case by case. Therefore, an integrated analysis strategy fusing several NiEEQ methods should be urgently developed and confirmed by proper clinical trials.

Bi-Directional Buck-Boost Forward Converter for Photovoltaic Module type Power Conditioning System (태양광 모듈형 전력조절기를 위한 양방향 벅-부스트 포워드 컨버터)

  • Kim, Kyoung-Tak;Jeon, Young-Tae;Park, Joung-Hu
    • The Transactions of the Korean Institute of Power Electronics
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    • v.21 no.4
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    • pp.335-342
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    • 2016
  • This paper proposes an energy storage-assisted, series-connected module-integrated power conversion system that integrates a photovoltaic power conditioner and a charge balancing circuit. In conventional methods, a photovoltaic power conditioner and a cell-balancing circuit are needed for photovoltaic systems with energy storage devices, but they cause a complex configuration and high cost. Moreover, an imbalanced output voltage of the module-integrated converter for PV panels can be a result of partial shading. Partial shading can lead to the fault condition of the boost converter in shaded modules and high voltage stresses on the devices in other modules. To overcome these problems, a bidirectional buck-boost converter with an integrated magnetic device operating for a charge-balancing circuit is proposed. The proposed circuit has multiple secondary rectifiers with inductors sharing a single magnetic core, which works as an inductor for the main bidirectional charger/discharger of the energy storage. The secondary rectifiers operate as a cell-balancing circuit for both energy storage and the series-connected multiple outputs of the module-integrated converter. The operating principle of the cell-balancing power conversion circuit and the power stage design are presented and validated by PSIM simulation for analysis. A hardware prototype with equivalent photovoltaic modules is implemented for verification. The results verify that the modularized photovoltaic power conversion system in the output series with an energy storage successfully works with the proposed low-cost bidirectional buck-boost converter comprising a single magnetic device.

Changes in pre-osteoblast cells associated with non-precious metal cores with dental implants: Pilot test (치과용 임플란트 적용 비귀금속 코어와 관련된 전조골세포의 변화)

  • Park, Jung-Hyun;Kang, Seen-Young;Kim, Jong-Woo;Kim, Jang-Ju;Kim, Woong-Chul;Kim, Ji-Hwan
    • Journal of Technologic Dentistry
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    • v.40 no.2
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    • pp.63-69
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    • 2018
  • Purpose: The purpose of this study is to investigate the non-precious metal core materials used in the dental laboratory to fabricate the implant superstructure by CAD / CAM method. And to observe and compare the morphology and distribution of the osteoblasts in relation to implant osseointegration. Methods: In this study, the mandibular right first molar tooth model was selected as an international standard to produce a single core. Using this model, the impression was made with the silicone rubber, the tooth model was scanned, and a single core was designed and 5-axis milling was performed. The materials used were Cobalt-Chromium and Nickel-Chromium, and the cores for dental implant top structures were fabricated according to the procedures of the dental labs. After the fabrication, the marginal area of the core was separated and cell culture experiment was performed. The osteoblast cells used MC3T3-E1, which is currently widely used. For morphological analysis of osteoblasts, cells were posttreated and observed using CLSM (Confocal Laser Scanning Microscope) and compared. Results: The cell adhesion behavior of the specimen surface measured by CLSM was uniformly distributed in specimen A (Cobalt-Chromium) than in specimen B (Nickel-Chromium). The distribution and changes of the cells were different in the two specimens. Conclusion : It is possible to confirm that specimen A (Cobalt-Chromium) is suitable for the living body through adhesion and proliferation of osteoblasts related to implant osseointegration in the non-precious metal superstructure used after implantation. It is considered that it is preferable to use Co-Cr when fabricating the superstructure.

Association of FOXP3 Single Nucleotide Polymorphisms With Clinical Outcomes After Allogenic Hematopoietic Stem Cell Transplantation

  • Nam, Minjeong;Shin, Sue;Park, Kyoung Un;Kim, Inho;Yoon, Sung-Soo;Kwon, Tack-Kyun;Song, Eun Young
    • Annals of Laboratory Medicine
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    • v.38 no.6
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    • pp.591-598
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    • 2018
  • Background: Forkhead box P3 (FOXP3) is an important marker of regulatory T cells. FOXP3 polymorphisms are associated with autoimmune diseases, cancers, and allograft outcomes. We examined whether single nucleotide polymorphisms (SNPs) at the FOXP3 locus are associated with clinical outcomes after allogenic hematopoietic stem cell transplantation (HSCT). Methods: Five FOXP3 SNPs (rs5902434, rs3761549, rs3761548, rs2232365, and rs2280883) were analyzed by PCR-sequencing of 172 DNA samples from allogenic HSCT patients. We examined the relationship between each SNP and the occurrence of graft-versus-host disease (GVHD), post-HSCT infection, relapse, and patient survival. Results: Patients with acute GVHD (grades II-IV) showed higher frequencies of the rs3761549 T/T genotype, rs5902434 ATT/ATT genotype, and rs2232365 G/G genotype than did patients without acute GVHD (P =0.017, odds ratio [OR]=5.3; P =0.031, OR=2.4; and P =0.023, OR=2.6, respectively). Multivariate analysis showed that the TT genotype of rs3761549 was an independent risk factor for occurrence of acute GVHD (P =0.032, hazard ratio=5.6). In contrast, the genotype frequencies of rs3761549 T/T, rs5902434 ATT/ATT, and rs2232365 G/G were lower in patients with post-HSCT infection than in patients without infection (P =0.026, P =0.046, and P =0.031, respectively). Conclusions: rs3761549, rs5902434, and rs2232365 are associated with an increased risk of acute GVHD and decreased risk of post-HSCT infection.

Optimized Methods of Preimplantation Genetic Diagnosis for Trinucleotide Repeat Diseases of Huntington's Disease, Spinocerebellar Ataxia 3 and Fragile X Syndrome (삼핵산 반복서열 질환인 헌팅톤병, 척수소뇌성 운동실조증, X-염색체 취약 증후군의 착상전 유전진단 방법에 대한 연구)

  • Kim, Min-Jee;Lee, Hyoung-Song;Lim, Chun-Kyu;Cho, Jae-Won;Kim, Jin-Young;Koong, Mi-Kyoung;Son, In-Ok;Kang, Inn-Soo;Jun, Jin-Hyon
    • Clinical and Experimental Reproductive Medicine
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    • v.34 no.3
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    • pp.179-188
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    • 2007
  • Objectives: Many neurological diseases are known to be caused by expansion of trinucleotide repeats (TNRs). It is hard to diagnose the alteration of TNRs with single cell level for preimplantation genetic diagnosis (PGD). In this study, we describe methods optimized for PGD of TNRs related diseases such as Huntington's disease (HD), spinocerebellar ataxia 3 (SCA3) and fragile X syndrome (FXS). Methods: We performed the preclinical assays with heterozygous patient's lymphocytes by single cell PCR strategy. Fluorescent semi-nested PCR and fragment analysis using automatic genetic analyzer were applied for HD and SCA 3. Whole genome amplification with multiple displacement amplification (MDA) method and fluorescent PCR were carried out for FXS. Amplification and allele drop-out (ADO) rate were evaluated in each case. Results: The fluorescent semi-nested PCR of single lymphocyte showed 100.0% of amplification and 14.0% of ADO rate in HD, and 94.7% of amplification and 5.6% of ADO rate in SCA3, respectively. We could not detect the PCR product of CGG repeats in FXS using the fluorescent semi-nested PCR alone. After applying the MDA method in FXS, 84.2% of amplification and 31.3% of ADO rate were achieved. Conclusions: Fluorescent semi-nested PCR is a reliable method for PGD of HD and SCA3. The advanced MDA method overcomes the problem of amplification failure in CGG repeats of FXS case. Optimization of methods for single cell analysis could improve the sensitivity and reliability of PGD for complicated single gene disorders of TNRs.

Analysis of Case Report Related with Mountain Ginseng Pharmacopuncture in Korean Medical Database (한국 논문 데이터베이스의 산삼약침 암 증례 논문 분석)

  • Park, Soo-jung;Joo, Jong-cheon
    • Journal of Korean Traditional Oncology
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    • v.20 no.2
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    • pp.51-59
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    • 2015
  • Objective: This paper is to investigate the current evidence and clinical effectiveness of mountain ginseng pharmacopuncture (MGP) in cancer treatment. Methods: Along with hand searches, relevant literatures were searched from Korean medical database until November 2015 without language restriction and clinical researches were selected for this review. Results: Nine articles were searched. The MGP was generally prepared in the concentration of ten percent. Also, the MGP has been used by intravenous injection much more than acupoint injection. The clinical effects were the suppression of the cancer cell growth, the improvement the quality of life, the reduction of the side effect by chemotherapy and radiotherapy. Conclusion: This analysis suggests that the MGP can be used as a single therapy or a supportive therapy with conventional therapies, which are chemotherapy or radiotherapy.