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Department of Biological SciencesDNA molecules are damaged by malfunctioning metabolism and bulky adducts, inducing genetic instability and passing it on to the next generation. DNA damage repair processes should be conducted properly. Thus, understanding the detail molecular mechanism of DNA damage repair process is important. My research focuses on DNA damage repair at the single molecular level, especially the dynamics of DNA damage recognition proteins on DNA. Chapter 1 Global genome nucleotide excision repair (GG-NER) eliminates chemical bulky adducts and UV-induced thymine dimers. This process is initiated by Xeroderma pigmentosum complementation group C protein (XPC), which detects DNA lesions by recognizing structural distortions. However, the exact mechanism for this detection and the factors affecting it are unclear. The detailed mechanism of XPC-RAD23B was revealed by DNA curtain assay, a single-molecule imaging technique that unidirectionally aligns DNA strands. The assay uses the fluidity of the lipid bilayer to track the movement of a single protein molecule on DNA in real time. I observed XPC-RAD23B (Red) jump over the protein obstacles (EcoRI^E111Q, Green) at specific sites while diffusing. Its diffusion coefficient increases in direct proportion to the ionic strength of the environment. These results indicate that human XPC-RAD23B uses diffusion along the DNA, especially via hopping, as a proxy for DNA lesions. This process allows it to bypass protein obstacles. Moreover, XPC-RAD23B moves along DNA strands in a heterogeneous fashion ??? at times immobile, diffusive, or constrained state, depending on the stability of DNA duplexes. Taken together, these results provide insight into how hXPC-Rad23B can rapidly find DNA defects, preventing mutations and ultimately cancer in human cells. Chapter 2 R-loops ??? three-stranded nucleotide structures consisting of an RNA-DNA hybrid and a displaced ssDNA structure ??? serve as signaling molecules in various cellular processes, but they also act as DNA lesions when improperly regulated. These molecules can be recognized and eliminated by Tonicity enhancer-binding protein (TonEBP), a transcription factor for immune response and tonicity regulation. Using a DNA curtain with purified TonEBP and R-loop-containing lambda DNA, I demonstrated that TonEBP identifies R-loops via both 1D diffusion and 3D collision. In addition, TonEBP preferentially binds the displaced ssDNA in the R-loop structure, as I confirmed using electrophoretic mobility shift assay with diverse types of DNA constructs. This study reveals that TonEBP recognizes R-loops on DNA and recruits R-loop elimination proteins such as METTL3-METTL14 to resolve the R-loops by RNase H1. Chapter 3 Although DNA curtain is a well-established single-molecule imaging technique, it???s imaging quality and experimental efficiency is compromised by two factors. First, on the chromium nano-barrier slide, the remaining materials for the DNA curtain assembly on the surface can induce protein aggregation, degrading the barriers in the harsh chemical and physical cleaning method. Second, in the DNA curtain, the aligned DNA molecules are visualized using YOYO-1, which causes rapid photocleavage of DNA under continuous laser illumination. We developed nano-trench, a novel type of DNA curtain barrier featuring an engraved zigzag pattern on the fused silica surface. Nano-trench is more durable and then conventional imaging techniques under harsh chemical treatment and causes less laser scattering. These features increase the quality of DNA curtain imaging. We also developed the FP-DBP, a chimeric construct of fluorescent protein (FP) and DNA binding peptide (DBP) that is less phototoxic than YOYO-1.ope

Biophysical and Biochemical Approaches for R-Loop Sensing Mechanism

An R-loop is a triple-stranded nucleic acid structure consisting of a DNA–RNA hybrid and a displaced single-stranded DNA. R-loops are associated with diverse biological reactions, such as immune responses and gene regulation, and dysregulated R-loops can cause genomic instability and replication stress. Therefore, investigating the formation, regulation, and elimination of R-loops is important for understanding the molecular mechanisms underlying biological processes and diseases related to R-loops. Existing research has primarily focused on R-loop detection. In this chapter, we introduce a variety of biochemical and biophysical techniques for R-loop sensing and visualization both in vivo and in vitro, including single-molecule imaging. These methods can be used to investigate molecular mechanisms underlying R-loop search and identification

Hair transplantation in patients with hair loss or scar deformity in the side hairline after midfacelifting surgery

Background Successful aesthetic plastic surgery is devoid of both unsightly scarring and postoperative disfigurement. Patients undergoing midface-lifting surgery are very often disconcerted by an altered side hairline, including sideburns, despite considerable amelioration of facial wrinkles. This study was conducted to identify an effective means of approaching an altered hairline and the unavoidable scarring arising from midface-lifting surgery. Methods A total of 37 patients who underwent corrective surgery with hair transplantation for hair loss or scar deformity arising from midface-lifting surgery from June 2014 to June 2017, and were observed for more than 6 months thereafter, were enrolled in the study. Prior to corrective surgery, the patients were administered a multiple-choice survey regarding their dissatisfaction arising from midface-lifting surgery. Among the 37 patients, 24, 12, and one underwent donor harvesting by the strip method, non-shaven follicular unit extraction, and partial shaving follicular unit extraction, respectively. Additionally, 33 of the 37 patients underwent hair transplantation in the frontotemporal recess area along with hairline correction surgery. The average number of transplanted grafts was 1,025. Results Surgery resulted in a natural and satisfactory appearance in all patients. The average patient and physician subjective satisfaction scores were 4.6 and 4.8, respectively. No adverse events such as folliculitis occurred. Conclusions Side-hairline correction surgery by hair transplantation can be considered an effective method of realigning an altered hairline accompanied by scars following midfacelifting surgery

Comparison of postoperative pain according to the harvesting method used in hair restorative surgery

Background Postoperative pain is one of the most common concerns of patients undergoing hair transplantation surgery. Because most patients are satisfied with the cosmetic improvement after transplantation, amelioration of postoperative pain would help to increase patient accessibility to hair restorative surgery and greatly impact patient satisfaction with the final cosmetic results. This study was performed to investigate postoperative pain after hair transplantation. Methods In total, 241 patients (202 who underwent follicular unit transplantation [FUT] and 39 who underwent follicular unit extraction [FUE]) were eligible for the study. Postoperative pain was evaluated on postoperative days 1, 2, 3, 4, 5, and 7 using the Wong-Baker Faces Pain Scale. The patients’ medical records were retrospectively reviewed for information on the harvesting method, number of transplanted grafts, size of donor design, and laxity, elasticity, and glidability of the scalp in relation to postoperative pain. Results Postoperative pain after hair transplantation, assessed with the Wong-Baker Faces Pain Scale, seemed to provide very subjective results. None of the variables were correlated with postoperative pain in the FUT group. Such pain, however, tended to disappear by postoperative day 3. Patients in the FUE group experienced significantly less severe pain than those in the FUT group. Conclusions Postoperative pain was significantly less severe in patients whose donor hair was harvested by the FUE than FUT method. Postoperative pain had almost disappeared by postoperative day 3 in the FUT group, whereas only minimal pain was present even on postoperative day 1 in the FUE group

99mTc-MAA accumulation within tumor in preoperative lung perfusion SPECT/CT associated with occult lymph node metastasis in patients with clinically N0 non-small cell lung cancer

Background 99mTc-MAA accumulation within the tumor representing pulmonary arterial perfusion, which is variable and may have a clinical significance. We evaluated the prognostic significance of 99mTc-MAA distribution within the tumor in non-small cell lung cancer (NSCLC) patients in terms of detecting occult nodal metastasis and lymphovascular invasion, as well as predicting the recurrence-free survival (RFS). Methods Two hundred thirty-nine NSCLC patients with clinical N0 status who underwent preoperative lung perfusion SPECT/CT were retrospectively evaluated and classified according to the visual grading of 99mTc-MAA accumulation in the tumor. Visual grade was compared with the quantitative parameter, standardized tumor to lung ratio (TLR). The predictive value of 99mTc-MAA accumulation with occult nodal metastasis, lymphovascular invasion, and RFS was assessed. Results Eighty-nine (37.2%) patients showed 99mTc-MAA accumulation and 150 (62.8%) patients showed the defect on 99mTc-MAA SPECT/CT. Among the accumulation group, 45 (50.5%) were classified as grade 1, 40 (44.9%) were grade 2, and 4 (4.5%) were grade 3. TLR gradually and significantly increased from grade 0 (0.009 ± 0.005) to grade 1 (0.021 ± 0.005, P < 0.05) and to grade 2–3 (0.033 ± 0.013, P < 0.05). The following factors were significant predictors for occult nodal metastasis in univariate analysis: central location, histology different from adenocarcinoma, tumor size greater than 3cm representing clinical T2 or higher, and the absence of 99mTc-MAA accumulation within the tumor. Defect in the lung perfusion SPECT/CT remained significant at the multivariate analysis (Odd ratio 3.25, 95%CI [1.24 to 8.48], p = 0.016). With a median follow-up of 31.5 months, the RFS was significantly shorter in the defect group (p = 0.008). Univariate analysis revealed that cell type of non-adenocarcinoma, clinical stage II-III, pathologic stage II-III, age greater than 65 years, and the 99mTc-MAA defect within tumor as significant predictors for shorter RFS. However, only the pathologic stage remained statistically significant, in multivariate analysis. Conclusion The absence of 99mTc-MAA accumulation within the tumor in preoperative lung perfusion SPECT/CT represents an independent risk factor for occult nodal metastasis and is relevant as a poor prognostic factor in clinically N0 NSCLC patients. 99mTc-MAA tumor distribution may serve as a new imaging biomarker reflecting tumor vasculatures and perfusion which can be associated with tumor biology and prognosis.This research was supported by the National Research Foundation of Korea (NRF) and funded by the Korean government (MSIT) (No.2020M3A9B6038086

Tumor immune profiles noninvasively estimated by FDG PET with deep learning correlate with immunotherapy response in lung adenocarcinoma

Rationale: The clinical application of biomarkers reflecting tumor immune microenvironment is hurdled by the invasiveness of obtaining tissues despite its importance in immunotherapy. We developed a deep learning-based biomarker which noninvasively estimates a tumor immune profile with fluorodeoxyglucose positron emission tomography (FDG-PET) in lung adenocarcinoma (LUAD). Methods: A deep learning model to predict cytolytic activity score (CytAct) using semi-automatically segmented tumors on FDG-PET trained by a publicly available dataset paired with tissue RNA sequencing (n = 93). This model was validated in two independent cohorts of LUAD: SNUH (n = 43) and The Cancer Genome Atlas (TCGA) cohort (n = 16). The model was applied to the immune checkpoint blockade (ICB) cohort, which consists of patients with metastatic LUAD who underwent ICB treatment (n = 29). Results: The predicted CytAct showed a positive correlation with CytAct of RNA sequencing in validation cohorts (Spearman rho = 0.32, p = 0.04 in SNUH cohort; spearman rho = 0.47, p = 0.07 in TCGA cohort). In ICB cohort, the higher predicted CytAct of individual lesion was associated with more decrement in tumor size after ICB treatment (Spearman rho = -0.54, p < 0.001). Higher minimum predicted CytAct in each patient associated with significantly prolonged progression free survival and overall survival (Hazard ratio 0.25, p = 0.001 and 0.18, p = 0.004, respectively). In patients with multiple lesions, ICB responders had significantly lower variance of predicted CytActs (p = 0.005). Conclusion: The deep learning model that predicts CytAct using FDG-PET of LUAD was validated in independent cohorts. Our approach may be used to noninvasively assess an immune profile and predict outcomes of LUAD patients treated with ICB.

Thyroid-Related Protein Expression in the Human Thymus

Radioiodine whole body scan (WBS), related to sodium iodide symporter (NIS) function, is widely used to detect recurrence/metastasis in postoperative patients with thyroid cancer. However, the normal thymic uptake of radioiodine has occasionally been observed in young patients. We evaluated the expression of thyroid-related genes and proteins in the human thymus. Thymic tissues were obtained from 22 patients with thyroid cancer patients of all ages. The expression of NIS, thyroid-stimulating hormone receptor (TSHR), thyroperoxidase (TPO), and thyroglobulin (Tg) was investigated using immunohistochemistry and quantitative RT-PCR. NIS and TSHR were expressed in 18 (81.8%) and 19 samples (86.4%), respectively, whereas TPO was expressed in five samples (22.7%). Three thyroid-related proteins were localized to Hassall’s corpuscles and thymocytes. In contrast, Tg was detected in a single patient (4.5%) localized to vascular endothelial cells. The expression of thyroid-related proteins was not increased in young thymic tissues compared to that in old thymic tissues. In conclusion, the expression of NIS and TSHR was detected in the majority of normal thymus samples, whereas that of TPO was detected less frequently, and that of Tg was detected rarely. The increased thymic uptake of radioiodine in young patients is not due to the increased expression of NIS

Single-molecule visualization reveals the damage search mechanism for the human NER protein XPC-RAD23B

DNA repair is critical for maintaining genomic integrity. Finding DNA lesions initiates the entire repair process. In human nucleotide excision repair (NER), XPC-RAD23B recognizes DNA lesions and recruits downstream factors. Although previous studies revealed the molecular features of damage identification by the yeast orthologs Rad4-Rad23, the dynamic mechanisms by which human XPC-RAD23B recognizes DNA defects have remained elusive. Here, we directly visualized the motion of XPC-RAD23B on undamaged and lesion-containing DNA using high-throughput single-molecule imaging. We observed three types of one-dimensional motion of XPC-RAD23B along DNA: diffusive, immobile and constrained. We found that consecutive AT-tracks led to increase in proteins with constrained motion. The diffusion coefficient dramatically increased according to ionic strength, suggesting that XPC-RAD23B diffuses along DNA via hopping, allowing XPC-RAD23B to bypass protein obstacles during the search for DNA damage. We also examined how XPC-RAD23B identifies cyclobutane pyrimidine dimers (CPDs) during diffusion. XPC-RAD23B makes futile attempts to bind to CPDs, consistent with low CPD recognition efficiency. Moreover, XPC-RAD23B binds CPDs in biphasic states, stable for lesion recognition and transient for lesion interrogation. Taken together, our results provide new insight into how XPC-RAD23B searches for DNA lesions in billions of base pairs in human genome

Enterovirus 71-associated hand, foot and mouth diseases with neurologic symptoms, a university hospital experience in Korea, 2009

PurposeHand-foot-mouth disease (HFMD) is a common viral illness in children, which is usually mild and self-limiting. However, in recent epidemics of HFMD in Asia, enterovirus 71 (EV71) has been recognized as a causative agent with severe neurological symptoms with or without cardiopulmonary involvement. HFMD was epidemic in Korea in the spring of 2009. Severe cases with complications including death have been reported. The clinical characteristics in children with neurologic manifestations of EV71 were studied in Ewha Womans University Mokdong Hospital.MethodsExaminations for EV71 were performed from the stools, respiratory secretion or CSF of children who presented neurologic symptoms associated with HFMD by realtime PCR. Clinical and radiologic data of the patients were collected and analyzed.ResultsEV71 was isolated from the stool of 16 patients but not from respiratory secretion or CSF. Among the 16 patients, meningitis (n=10) was the most common manifestation, followed by Guillain-Barré syndrome (n=3), meningoencephalitis (n=2), poliomyelitis-like paralytic disease (n=1), and myoclonus (n=1). Gene analysis showed that most of them were caused by EV71 subgenotype C4a, which was prevalent in China in 2008.ConclusionBecause EV71 causes severe complications and death in children, a surveillance system to predict upcoming outbreaks should be established and maintained and adequate public health measures are needed to control disease