30Kc6 유전자를 이용한 역분화줄기세포의 세포자멸 억제 효과 및 세포의 직접변환을 위한 30Kc19 단백질 기반 전사인자의 효율적인 전달

학위논문 (박사)-- 서울대학교 대학원 공과대학 협동과정 바이오엔지니어링전공, 2017. 8. 황석연.30K protein, derived from silkworm hemolymph (SH), was known as having many properties. From 30K family (30Kc6, 30Kc12, 30Kc19, 30Kc21, and 30Kc23), 30Kc6 plays role for anti-apoptosis. When the cells were transfected with 30Kc6, the cells had higher viability in stress condition. Also, these property of 30Kc6 could increase the productivity of antibody, recombinant interferon-β, and human erythropoietin (EPO). The mechanism of 30Kc6 for antiapoptosis was due to prevent Bax binding on mitochondria. The other 30K protein, 30Kc19, has reported the properties of cell-penetrating and enzyme-stabilizing. Cell-penetrating peptide (Pep-c19) of 30Kc19 could enter cells by forming dimer. Also, 30Kc19 stabilized enzyme by crowd effect. Recent, 30Kc19 protein was reported enhancing soluble expression for transcription factors. The objective of this research is to overcome several hurdles in stem cell research. Human pluripotent stem cells such as hES and hiPS cells suffer from apoptosis in single cell-dissociation. Because of pre-activated Bax at Golgi in cells, hES and hiPS cells induced rapid apoptosis in stress condition. We applied 30Kc6 in hiPS cells. hiPS-30Kc6 cells were expressed pluripotent stem cell markers, and had a potency of differentiation in three germ layers. Also, the viability of single cells was higher than normal hiPS cells. Then, we wondered that anti-apoptotic property of 30Kc6 was from a specific domain of 30Kc6. From the apoptosis research, BH4 domain of Bcl2 inhibited apoptosis by binding Bax. With structural similarity of BH4, 30Kc6 alpha-helix (30Kc6α) was considered as anti-apoptotic domain. By truncation, we cloned 30Kc6α and observed anti-apoptotic property. Furthermore, 30Kc19-30Kc6α protein could penetrate cell, and showed enhanced cellular viability under STS or UV-irradiation condition. Cell-penetration and enzyme-stabilization are the properties on 30Kc19 protein. We tried to solve the problems on reprogramming which are instability and low solubility of transcription factor proteins. Previous reprogramming methods had a risk of transgene integration to host genome. By using protein, transcription factors were delivered directly without any DNA-related complications. To express transcription factors as soluble form, 30Kc19 was conjugated. 30Kc19 conjugated transcription factors (Oct4, Sox2, c-Myc, and Klf4) were expressed as a soluble protein. Purified soluble transcription factors were penetrated into cells, and stable up to 48 h. Furthermore, Klf4-30Kc19 protein showed a transcriptional activity. Lastly, we generated neuronal cells using transcription factor with 30Kc19. 30Kc19-Ascl1 protein induced successfully mouse embryonic fibroblasts (MEFs) to protein-induced neuronal cells (piNCs). p-iNCs had neuronal morphology, and expressed neuronal markers (Tuj1 and MAP2). Furthermore, the expression levels of neuronal genes (ASCL1, BRN2, and MYT1L) were observed higher on day 7 and 14. In this research, we applied anti-apoptotic 30Kc6 and cellpenetrating 30Kc19 protein on stem cell research. This approach will give a chance for developing new techniques in stem cell research.Chapter 1. Research background and objective ................................. 1 Chapter 2. Literature review ............................................................... 6 2.1 Cellular reprogramming .......................................................... 7 2.2 Reprogramming methods ........................................................ 9 2.2.1 Virus .................................................................................. 9 2.2.2 DNA ..................................................................................10 2.2.3 mRNA and miRNA .......................................................... 13 2.2.4 Cre/LoxP and PiggyBac transposon .............................. 14 2.2.5 Protein ............................................................................. 16 Chapter 3. Experimental procedures ................................................ 18 3.1 Gene cloning .......................................................................... 19 3.2 Protein production and analysis ........................................... 19 3.2.1 Protein expression and purification …............................ 19 3.2.2 Quantitative analysis using BSA standard solution ...... 20 3.2.3 Western blot analysis for in vitro stability assay ......... 21 3.3 Cell culture ..........................................................................… 22 3.3.1 Primary cell isolation and culture .................................. 22 3.3.2 Embryonic stem cell culture and embryoid body formation ............................................................................ 23 3.4 Generation of induced pluripotent stem cell (iPSC) ......…… 24 3.4.1 Retrovirus production ..................................................... 24 3.4.2 Viral transduction and iPSC culture ............................... 24 3.5 Generation of protein-induced neuronal cell (p-iNC) ....... 27 3.6 Cell analysis ........................................................................... 27 3.6.1 RNA isolation and cDNA synthesis ............................... 27 3.6.2 RT-PCR and RT-qPCR ................................................. 28 3.6.3 Immunocytochemistry .................................................... 31 3.6.4 Live cell analysis ............................................................ 31 3.6.5 Cell viability assay .......................................................... 32 3.6.6 Luciferase assay ............................................................ 32 3.6.7 Apoptosis induction and FACS analysis ........................ 33 3.6.8 Single cell-dissociation and Alkaline phosphatase staining .............................................................................. 34 3.6.9 Electrophysiological recording ………............................... 35 Chapter 4. Anti-apoptotic effect of 30Kc6 gene on human induced pluripotent stem cell ........................................................................... 36 4.1 Introduction ........................................................................... 37 4.2 Retroviral transduction and expression of hESC-specific genes in hiPSC-30Kc6 ........................................................ 40 4.3 In vitro differentiation of hiPSC-30Kc6 ................................. 44 4.4 Anti-apoptosis effect on hiPS-30Kc6 cells ....................... 46 4.5 Single cell-dissociated cell viability .................................... 49 4.6 Conclusions ............................................................................. 52 Chapter 5. Anti-apoptotic role of 30Kc6 alpha helix domain and its application by conjugating with 30Kc19 protein ............................... 53 5.1 Introduction ............................................................................ 54 5.2 Cloning of 30Kc6α and gene expression in mammalian cells ...................................................................... 57 5.3 Anti-apoptosis property of 30Kc6α .................................. 59 5.4 Expression and purification of 30Kc19-30Kc6α protein ............................................................................................ 62 5.5 Cytotoxicity and cell penetration of 30Kc19-30Kc6α protein ................................................................................... 62 5.6 Anti-apoptosis property of 30Kc19-30Kc6α protein ..... 63 5.7 Conclusions ………………………..……………………….……………………….…. 67 Chapter 6. Soluble expression and stability enhancement of transcription factors using 30Kc19 cell-penetrating protein .................................................................................................. 69 6.1 Introduction ………………………………………………………….….……………… 70 6.2 Soluble expression and purification of transcription factors ………………………………………………………………………….…………... 74 6.3 Analysis of purified soluble transcription factors ............... 78 6.4 In vitro stability of soluble transcription factors ................ 78 6.5 Cell penetration and intracellular stability of soluble transcription factors ………………………..................................... 82 6.6 Cytotoxicity of 30Kc19-conjugated transcription factors ........................................................................................... 83 6.7 Transcriptional activity of 30Kc19-conjugated Klf4 ........ 86 6.8 Conclusions ........................................................................... 87 Chapter 7. Direct conversion of fibroblasts to neuronal cells by 30Kc19-Ascl1-NLS-R9 protein ..................................................... 91 7.1 Introduction ………………………………………………………….…….………….. 92 7.2 Soluble expression of 30Kc19-Ascl1-NLS-R9 protein ........................................................................................... 93 7.3 Purification, and in vitro stability of protein …………….……….. 96 7.4 Cell penetration ……………………………………………………….…...…..….. 98 7.5 Cytotoxicity assay ………………………………………………………….…… 100 7.6 Generation of protein-induced neuronal cells (p-iNCs) ......................................................................................... 100 7.7 Cell morphology change of p-iNCs …………………………...……. 103 7.8 Expression of neuronal biomerkers in p-iNCs ……….……… 103 7.9 Neuronal gene expression in p-iNCs …………………….…..…... 106 7.10 Electrophysiological recordings …………………….…..………..... 106 7.11 Conclusions ………………………………………………………..………...…… 110 Chapter 8. Overall discussion and further suggestions ................. 112 Bibliography ...................................................................................... 119 Korean abstract ................................................................................ 132Docto

Role of Thromboelastography as an Early Predictor of Disseminated Intravascular Coagulation in Patients with Septic Shock

(1) Background: The currently proposed criteria for diagnosing overt disseminated intravascular coagulation (DIC) are not suitable for early detection of DIC. Thromboelastography (TEG) rapidly provides a comprehensive assessment of the entire coagulation process and is helpful as a guide for correcting consumptive coagulopathy in sepsis-induced DIC. This study aimed to investigate the role of TEG in the prediction of DIC in patients with septic shock. (2) Methods: TEG was conducted prospectively in 1294 patients with septic shock at the emergency department (ED) between January 2016 and December 2019. After exclusion of 405 patients with "do not attempt resuscitation" orders, those refusing enrollment, and those developing septic shock after ED presentation, 889 patients were included. DIC was defined as an International Society on Thrombosis and Hemostasis score >= 5 points within 24 h. (3) Results: Of the 889 patients with septic shock (mean age 65.6 +/- 12.7 years, 58.6% male), 158 (17.8%) developed DIC. TEG values, except lysis after 30 min, were significantly different between the DIC and non-DIC groups. Among the TEG values, the maximal amplitude (MA) had the highest discriminating power for DIC, with an area under the curve of 0.814. An MA < 60 indicated DIC with 79% sensitivity, 73% specificity, and 94% negative predictive value. Based on multivariable analysis, MA < 60 was an independent predictor of DIC (odds ratio 5.616 (95% confidence interval: 3.213-9.818)). (4) Conclusions: In patients with septic shock, the MA value in TEG could be a valuable tool for early prediction of DIC.ope

Simulation study: the development of a respiratory barrier enclosure with negative pressure and the analysis of its protective effect during intubation

Objective: Within the last 2 years, coronavirus disease 2019 has spread rapidly across several continents, with 100 million confirmed infected patients. Physical barrier enclosure, also called “aerosol-box,” is a solution for the shortage of protective devices and spaces. In this study, we examined the safety of the novel barrier enclosure. Methods: We simulated droplets by nebulizing 1% glycerol+99% ethanol solution. Two experienced physicians performed intubation under two conditions, such as the isolator condition (applying isolator without negative condition) and the negative pressure condition (applying isolator with the negative condition). We compared two conditions with two control groups, including negative control (room air) and positive control (synthetizing droplet air). During the procedure, particles were counted for 30 seconds, and this was repeated 10 times. At each condition, depending on the result of the normality test (Shapiro-Wilk test), an independent t-test was used when normality was satisfied, and a Mann-Whitney Utest was used when normality was not satisfied. Results: The total number of particles in the positive control was 308,788 (175,936-461,124). The total number of particles for both conditions was significantly less than the positive control. Total number of particles in the isolator condition was 30,952 (27,592-33,244, P=0.001) and that in the negative pressure condition was 27,890 (27,165-29,786, P=0.001). Conclusion: The novel barrier significantly reduces synthetizing droplets exposure during intubation. Application of negative pressure through the isolator results in an additional decrease in particle exposure. Studies involving a larger population of operators and prolonged procedures are required.ope

The Usefulness of Non-face-to-face Communication Device for Medical Consents in the Emergency Department During COVID-19 Pandemic

Purpose : Since the era of COVID-19, face-to-face contact has been reduced to prevent the spread of infectious diseases around the world, and hospitals are applying various methods to prevent the spread ofinfection. However, when writing a consent form essential during the treatment process, it had to be done face-to-face. We developed a non-face-to-face communication device to enable real-time consent writing. This study aims to evaluate the usefulness of the non-face-to-face communication device when writing a consent form. Methods : From December 28, 2021 to February 2, 2022, electronic medical records of patients visited the severance hospital emergency center and had a central venous catheter inserted were collectedretrospectively. There were 56 consent forms included in the study, 43 face-to-face and 13 non-face-to-face. We checked the difference between the details explained in the non-face-to-face consent form and theface-to-face by the average score and the explanation of each item. The score was measured from a minimum of 0 points to a maximum of 13 points, with 1 point for explained items and 0 points forunexplained. Results : The average score for the face-to-face consent form was 4.3, and for the non-face-to-face was 3.0 (p=0.148). There was no significant difference in the explanation of each item. Conclusion : It was confirmed writing a consent form through the non-face-to-face communication device can perform a similar role compared to the face-to-face. It is expected unnecessary contact can be reducedby applying the device to hospital rooms, and enabling a non-face-to-face rounds system for new infectious diseases.ope

Prevalence of Carbon Monoxide Poisoning and Hyperbaric Oxygen Therapy in Korea: Analysis of National Claims Data in 2010-2019

This study aimed to investigate the prevalence of carbon monoxide (CO) poisoning and the provision of hyperbaric oxygen therapy (HBOT) in South Korea. We used data from the Korea Health Insurance Review and Assessment service. In total, 44,361 patients with CO poisoning were identified across 10 years (2010–2019). The prevalence of CO poisoning was found to be 8.64/10,000 people, with a gradual annual increment. The highest prevalence was 11.01/10,000 individuals, among those aged 30–39 years. In 2010, HBOT was claimed from 15 hospitals, and increased to 30 hospitals in 2019. A total of 4,473 patients received HBOT in 10 years and 2,684 (60%) were treated for more than 2 hours. This study suggested that the prevalence of both CO poisoning and HBOT in Korea gradually increased over the past 10 years, and disparities in prevalence were observed by region.ope

Risk factors to predict post-contrast acute kidney injury after contrast-enhanced computed tomography in the emergency department

Objective: This study aimed to investigate the risk factors of post-contrast acute kidney injury (PAKI) and the usefulness of the Mehran score for predicting PAKI in patients who underwent contrast-enhanced abdominopelvic computed tomog raphy (CE-APCT) in the emergency department (ED). Methods: This was a retrospective observational study. Patients who underwent CE-APCT and had a follow-up creati nine test within 72 hours in the period January to June, 2017, were enrolled for the study. PAKI is defined as a 25% or higher increase in the level of serum creatinine (sCr) within 72 hours after receiving contrast, or an increase in the level of sCr by 0.5 mg/dL. The odds ratio (OR) of risk factors and incidence of PAKI after CE-APCT were analyzed according to the Mehran risk group, and compared to expected incidence. Univariate and multivariate logistic regression analyses were performed for each risk factor. Results: A total of 1,718 patients were enrolled in the study. Of these, 203 patients (11.8%) developed PAKI, and 2 patients (0.1%) required dialysis. Hypotension (systolic blood pressure <80 mmHg) was determined to be statistically sig nificant (P=0.029; OR, 3.181) among the considered risk factors of PAKI. In the group having abnormal estimated glomerular filtration rate (<90 mL/min/1.73 m2 ), the age and rate of the underlying disease (congestive heart failure, hypertension) was found to be higher in the PAKI group. The receiver operating curve of Mehran score (area under the curve: 0.521 in model A, 0.520 in model B) was statistically not significant in the univariate analysis. A higher Mehran score was associated with a higher proportion of patients who underwent prophylactic treatment. Conclusion: There are no definite useful risk factors, including the Mehran score, for predicting PAKI in patients who underwent contrast-enhanced computed tomography in the EDope

Comparison of the survival and neurological outcomes in OHCA based on smoking status: investigation of the existence of the smoker's paradox

The smoker’s paradox has been reported to reduce mortality following out-of-hospital cardiac arrest (OHCA). However, recent studies on this paradox have reported contradictory findings, with some indicating that it does not exist. Therefore, the purpose of this study was to evaluate the association between smoking status and OHCA outcomes. This retrospective observational study was conducted using multicenter registry data. The associations between smoking status and OHCA outcomes were assessed using multivariable logistic regression analyses and propensity score-adjusted methods. We compared outcomes among current, former, and never-smokers, as well as between current and non-smokers and between ever- and never-smokers. The primary outcome was survival to hospital discharge, and the secondary outcome was favourable neurological outcomes. Among 4443 patients with OHCA, 19.9% were current smokers, 15.2% were former smokers, and 64.9% were never-smokers. Current smokers had significantly better outcomes than former or never-smokers. However, the significant differences observed in univariable analysis or before propensity score matching were not observed after adjustments with multivariable logistic regression or after propensity score matching analysis in both current vs. non-smokers and ever- vs. never-smokers. Other propensity score adjusted models also did not show significant differences, except for the stratification method. This study suggests that smoking is not an independent prognostic factor for OHCA. The statistically significant better outcomes observed in current or ever-smokers were not maintained after adjusting for confounders. Therefore, the smoker’s paradox should be investigated in additional prospective studies.ope

Systematic review for economic benefit of poison control center

Purpose: The purpose of this study was to conduct a systematic review to investigate the socio-economic benefits of the poison control center (PCC) and to assess whether telephone counseling at the poison control center affects the frequency of emergency room visits, hospitalization, and length of stay of patients with acute poisoning. Methods: The authors conducted a medical literature search of the PubMed, EMBASE, and Cochrane Library databases. Two reviewers evaluated the abstracts for eligibility, extracted the data, and assessed the study quality using a standardized tool. Key results such as the cost-benefit ratio, hospital stay days, unnecessary emergency room visits or hospitalizations, and reduced hos pital charges were extracted from the studies. When meta-analysis was possible, it was performed using RevMan software (RevMan version 5.4). Results: Among 299 non-duplicated studies, 19 were relevant to the study questions. The cost-benefit ratios of PCC showed a wide range from 0.76 to 36 (average 6.8) according to the level of the medical expense of each country and whether the study included intentional poisoning. PCC reduced unnecessary visits to healthcare facilities. PCC consultation shortened the length of hospital stay by 1.82 (95% CI, 1.07-2.57) days. Conclusion: The systematic review and meta-analysis support the hypothesis that the PCC operation is cost-beneficial. However, when implementing the PCC concept in Korea in the future, it is necessary to prepare an institutional framework to ensure a cost effective model.ope

Prognostic value of repeated thromboelastography measurement for favorable neurologic outcome during targeted temperature management in out-of-hospital cardiac arrest survivors

Background: Cardiac arrest can activate blood coagulation, which clinically manifests as obstruction of the microcirculation and multiple organ dysfunction. Thromboelastography (TEG) provides a rapid and comprehensive assessment of hemostatic processes, but there are limited data on the use of sequential TEG values during targeted temperature management (TTM) in out-of-hospital cardiac arrest (OHCA) survivors. The aim of this study was to investigate the prognostic value of coagulopathy assessed by repeated TEG to predict neurologically intact survival. Methods: A prospective cohort of consecutive non-trauma OHCA patients who were successfully resuscitated and treated with TTM. Patients with a target temperature of 36 ℃, no TEG data, and who declined appropriate treatment were excluded. TEG was measured at three time points of TTM (initial phase, target phase, and rewarming phase). The primary outcome was 28 day favorable neurologic function, defined as a Cerebral Performance Category of 1 or 2. Results: A total of 125 patients (mean age, 61 years; 63.2% male) were analyzed. A favorable neurologic outcome at 28 days was seen in 40 patients (32.0%). TEG values of R and LY30 in the initial phase were significantly lower in the favorable neurologic outcome group than in the unfavorable group (5.8 vs. 8.1 and 0.1 vs. 0.7, respectively; p 0.05 for all). Univariate analysis showed higher D-dimer levels, prothrombin time, and activated partial thromboplastin time in the unfavorable outcome group. In the multivariable analysis, TEG values of combination of R < 5 and LY30 < 7.5 in the initial phase were the only coagulation profiles seen to be independently associated with favorable neurologic outcome (OR, 4.508, 95% CI, 1.254-16.210). Conclusion: TEG results are available within minutes, and shorted R values or the absence of prolonged LY30 values in the initial phase are an early predictor of neurologically intact survival in successfully resuscitated OHCA patients.restrictio

Role of thromboelastography in the evaluation of septic shock patients with normal prothrombin time and activated partial thromboplastin time

Coagulopathy is frequent in septic shock and plays a key role in multiple organ dysfunction. The aim of this study is to investigate application values of thromboelastography (TEG) for outcome in septic shock patients with a normal value of prothrombin time (PT) and active partial thromboplastin time (aPTT). Prospective observational study using 1298 consecutive septic shock patients with TEG at admission was conducted at the emergency department (ED) of a tertiary care hospital in South Korea between 2016 and 2019. After excluding overt-disseminated intravascular coagulation (DIC) defined by scoring system, we included patients with a normal value of international normalized ratio ≤ 1.3 and aPTT ≤ 34 s. The primary outcome was 28-day mortality. 893 patients were included and 129 patients with overt DIC were excluded. Of the 764 remaining patients, 414 (54.2%) patients showed normal PT and aPTT (28-day mortality rate, 11.4%). TEG values such as reaction time, kinetic time (K), alpha angle (α), maximum amplitude (MA) and lysis index (LY 30) showed no significant mean difference between the survivor and non-survivor groups. However, hypocoagulable TEG values such as α 3 and α < 53 and MA < 50) was independent factors associated with increased risk of death (OR 4.882 [95% CI, 1.698-14.035]; p = 0.003). In conclusion, septic shock patients with normal PT and aPTT can be associated with impaired TEG profile, such as hypocoagulability, associated with increased mortality.ope