합성 생물학 분야의 플래폼 기술로써의 DNA 합성 이해

방두희 한국생물공학회 2012 BT NEWS Vol.19 No.2

Promiscuous Trans-splicing Activities Revealed by Next Generation Sequencing-based Analysis of 298 Split Inteins

서한나,방두희 한국생물공학회 2020 Biotechnology and Bioprocess Engineering Vol.25 No.2

Protein trans-splicing is a naturally occurring process in which two protein fragments are ligated by a reaction between two intein domains, called split inteins. Despite their usefulness in research, the reactivity and structure of only a few split inteins have been studied. We used cell-based kanamycin selection and next-generation sequencing (NGS) to simultaneously measure the splicing reactivity of 298 N-intein–C-intein combinations derived from the DnaE gene of cyanobacteria. Additionally, we confirmed the splicing activities by measuring the growth of cells individually harboring each split intein under kanamycin selection. Overall, the N-intein–C-intein combinations were promiscuous in their trans-splicing activities, although certain combinations did not splice actively. These results and the NGS-based analysis in this research would be helpful for the development of novel split inteins and further understanding of the trans-splicing mechanism.

Single-cell RNA sequencing technologies and bioinformatics pipelines

황병진,이지현,방두희 생화학분자생물학회 2018 Experimental and molecular medicine Vol.50 No.-

Rapid progress in the development of next-generation sequencing (NGS) technologies in recent years has provided many valuable insights into complex biological systems, ranging from cancer genomics to diverse microbial communities. NGS-based technologies for genomics, transcriptomics, and epigenomics are now increasingly focused on the characterization of individual cells. These single-cell analyses will allow researchers to uncover new and potentially unexpected biological discoveries relative to traditional profiling methods that assess bulk populations. Single-cell RNA sequencing (scRNA-seq), for example, can reveal complex and rare cell populations, uncover regulatory relationships between genes, and track the trajectories of distinct cell lineages in development. In this review, we will focus on technical challenges in single-cell isolation and library preparation and on computational analysis pipelines available for analyzing scRNA-seq data. Further technical improvements at the level of molecular and cell biology and in available bioinformatics tools will greatly facilitate both the basic science and medical applications of these sequencing technologies.

MAPS-seq: magnetic bead-assisted parallel single-cell gene expression profiling

Munsu Park,이동인,방두희,Ji Hyun Lee 생화학분자생물학회 2020 Experimental and molecular medicine Vol.52 No.-

Recently developed single-cell RNA sequencing methods allow the simultaneous profiling of the transcriptomes of thousands of individual cells. However, current methods still require advanced equipment or entail substantial waste of reagents. Here, we introduce magnetic bead-assisted parallel single-cell gene expression sequencing (MAPS-seq), a microwell-based method that pools samples before the reverse transcription step, increasing the ease of sample preparation and reducing reagent waste. Moreover, because this method uses universal reagents and standard molecular biology lab instruments, it is easy to implement, even in labs that have not previously conducted single-cell RNA sequencing. We validated our method by demonstrating that it can generate gene expression data at the singlecell level. We then applied the MAPS-seq method to analyze 237 human myelogenous leukemia cells treated with one of three different drugs or dimethyl sulfoxide. We observed transcriptional changes and identified marker genes that indicate a drug response. Furthermore, the MAPS-seq method produced data of comparable quality to those of existing single-cell RNA sequencing methods. Consequently, we expect that our method will provide researchers with a more accessible, less wasteful, and less burdensome method for investigating the transcriptomes of individual cells.

Native Chemical Ligation을 통한 티타늄 산화물 기판에의 폴리에틸렌글리콜 고정

변은경,김장배,강성민,이혁진,방두희,이해신 한국표면공학회 2012 한국표면공학회 학술발표회 초록집 Vol.2012 No.11

Clinical and Pathological Heterogeneity of Korean Patients with CAPN3 Mutations

최영철,박형준,장훈,이정환,신하영,조성래,박기덕,방두희,이민구,김승민,이지현 연세대학교의과대학 2016 Yonsei medical journal Vol.57 No.1

Purpose: This study was designed to investigate the characteristics of Korean patients with calpainopathy. Materials and Methods: Thirteen patients from ten unrelated families were diagnosed with calpainopathy via direct or targeted sequencing of the CAPN3 gene. Clinical, mutational, and pathological spectra were then analyzed. Results: Nine different mutations, including four novel mutations (NM_000070: c.1524+1G>T, c.1789_1790inA, c.2184+1G>T, and c.2384C>T) were identified. The median age at symptom onset was 22 (interquartile range: 15–28). Common clinical findings were joint contracture in nine patients, winged scapula in four, and lordosis in one. However, we also found highly variable clinical featuresincluding early onset joint contractures, asymptomatic hyperCKemia, and heterogeneous clinical severity in three members of the same family. Four of nine muscle specimens revealed lobulated fibers, but three showed normal skeletal muscle histology. Conclusion: We identified four novel CAPN3 mutations and demonstrated clinical and pathological heterogeneity in Korean patientswith calpainopathy.

NFATC3–PLA2G15 Fusion Transcript Identified by RNA Sequencing Promotes Tumor Invasion and Proliferation in Colorectal Cancer Cell Lines

장지은,김황필,한세원,장훈,이시현,송상현,방두희,김태유 대한암학회 2019 Cancer Research and Treatment Vol.51 No.1

Purpose This study was designed to identify novel fusion transcripts (FTs) and their functional significance in colorectal cancer (CRC) lines. Materials and Methods We performed paired-end RNA sequencing of 28 CRC cell lines. FT candidates were identified using TopHat-fusion, ChimeraScan, and FusionMap tools and further experimental validation was conducted through reverse transcription-polymerase chain reaction and Sanger sequencing. FT was depleted in human CRC line and the effects on cell proliferation, cell migration, and cell invasion were analyzed. Results One thousand three hundred eighty FT candidates were detected through bioinformatics filtering. We selected six candidate FTs, including four inter-chromosomal and two intrachromosomal FTs and each FT was found in at least one of the 28 cell lines. Moreover, when we tested 19 pairs of CRC tumor and adjacent normal tissue samples, NFATC3– PLA2G15 FT was found in two. Knockdown of NFATC3–PLA2G15 using siRNA reduced mRNA expression of epithelial–mesenchymal transition (EMT) markers such as vimentin, twist, and fibronectin and increased mesenchymal–epithelial transition markers of E-cadherin, claudin-1, and FOXC2 in colo-320 cell line harboring NFATC3–PLA2G15 FT. The NFATC3–PLA2G15 knockdown also inhibited invasion, colony formation capacity, and cell proliferation. Conclusion These results suggest that that NFATC3–PLA2G15 FTs may contribute to tumor progression by enhancing invasion by EMT and proliferation.

Efficacy of Olaparib in Treatment-Refractory, Metastatic Breast Cancer with Uncommon Somatic BRCA Mutations Detected in Circulating Tumor DNA

윤정기,안종성,김시현,김황필,강준규,방두희,임유주,김태유 대한암학회 2023 Cancer Research and Treatment Vol.55 No.3

Poly(ADP-ribose) polymerase inhibitors have been shown dramatic responses in patients with BRCAness. However, clinical studies have been limited to breast cancer patients with germline mutations. Here, we describe a patient with metastatic breast cancer who had a rare BRCA1 somatic mutation (BRCA1 c.4336G>T (p.E1446*)) detected by cell-free DNA analysis after failing standard therapies. This tier III variant of unknown significance was predicted to be a pathogenic variant in our assessment, leading us to consider off-label treatment with olaparib. The patient responded well to olaparib for several months, with a decrease in allele frequency of this BRCA1 somatic mutation in cell-free DNA. Olaparib resistance subsequently developed with an increase in the allele frequency and new BRCA1 reversion mutations. To our knowledge, this is the first report confirming BRCA1 c.4336G>T (p.E1446*) as a mutation sensitive to olaparib in breast cancer and describing the dynamic changes in the associated mutations using liquid biopsy.

The Impact of Cigarette Smoking on the Frequency of and Qualitative Differences in KRAS Mutations in Korean Patients with Lung Adenocarcinoma

김혜련,이진구,하상준,남기창,라선영,김주항,조병철,안정륜,방두희,홍윤경,김선미,Ross A. Soo,Gregory J. Riely 연세대학교의과대학 2013 Yonsei medical journal Vol.54 No.4

Purpose: This study was designed to determine the relationship of cigarette smoking to the frequency and qualitative differences among KRAS mutations in lung adenocarcinomas from Korean patients. Materials and Methods: Detailed smoking histories were obtained from 200 consecutively enrolled patients with lung adenocarcinoma according to a standard protocol. EGFR (exons 18 to 21) and KRAS (codons 12/13) mutations were determined via direct-sequencing. Results:The incidence of KRAS mutations was 8% (16 of 200) in patients with lung adenocarcinoma. KRAS mutations were found in 5.8% (7 of 120) of tumors from never-smokers, 15% (6 of 40) from former-smokers, and 7.5% (3 of 40) from current-smokers. The frequency of KRAS mutations did not differ significantly according to smoking history (p=0.435). Never-smokers were significantly more likely than former or current smokers to have a transition mutation (G→A or C→T) rather than a transversion mutation (G→T or G→C) that is known to be smoking-related (p=0.011). In a Cox regression model, the adjusted hazard ratios for the risk of progression with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) were 0.24 (95% CI, 0.14-0.42; p<0.001) for the EGFR mutation and 1.27 (95% CI, 0.58-2.79; p=0.537) for the KRAS mutation. Conclusion:Cigarette smoking did not influence the frequency of KRAS mutations in lung adenocarcinomas in Korean patients, but influenced qualitative differences in the KRAS mutations.