Discovery of anthrax biomarker candidates from human aortic endothelial cells : 인간 대동맥 내피세포 모델에서의 탄저 바이오마커 후보 발굴

조혜은 서울대학교 대학원 2014 국내석사

Anthrax, a zoonotic disease caused by Bacillus anthracis, is classified as cutaneous, gastrointestinal, or inhalational anthrax depending on the route of infection. The most threatening and fatal form is inhalational anthrax, which can be used as a biological weapon. B. anthracis releases three types of toxins, protective antigen, lethal factor, and edema factor that cause vascular and organ hemorrhage, and eventually fatal damage to the host. Since the early symptoms of inhalational anthrax are similar to those of the common cold, the status of infection is difficult to recognize. Moreover, any monitoring and prognostic tools have not been developed to predict recovery of anthrax patients. Therefore, infected patients receive late or unsuitable treatments, contributing to their increased mortality rate. Our purpose in this study was to identify the candidates of surrogate biomarker for anthrax that can be used to rapidly diagnose and monitor treatment outcomes. To evaluate the effects of anthrax toxins?lethal toxin (LT) and edema toxin (ET)?, cytotoxicity, cell growth inhibition, and cell morphology were analyzed in primary human aortic endothelial cells (HAECs). ET induced morphological changes in HAECs while maintaining cell viability similar to that of the control group. LT reduced cell viability in a concentration- and time-dependent manner. To discover the candidates of surrogate biomarker, secretome from anthrax toxin-treated HAECs was analyzed by using nano-liquid chromatography electrospray ionization tandem mass spectrometry. Proteins significantly upregulated or downregulated in comparison to the control group were selected, and four proteomic candidates were confirmed using immunoblot analysis. Therefore, lactotransferrin, pregnancy zone protein, and sarcolemmal membrane-associated protein were identified as proteomic candidates of anthrax biomarker. In addition, we analyzed exosomal small RNAs and miRNAs secreted from toxin-treated HAECs by using expression array and microarray to discover transcriptomic candidates. One miRNA and several small RNAs were selected and verified using real-time quantitative reverse transcription polymerase chain reaction. As a results, ASPH, CD151, HSP90AA1, ICAM2, LYPD1, MPST, NUAK1 and hsa-miR-1307-3p were found as transcriptomic candidates of anthrax biomarker. We intend to confirm the clinical utility of these proteomic and transcriptomic candidates as anthrax biomarker using sera obtained from rat injected with anthrax toxins. In this study, three proteins, seven small RNAs, and one miRNA were identified and validated in an in vitro anthrax model. These results suggested that secretory proteins and exosomal RNAs could be used as putative anthrax biomarkers for early diagnosis and treatment monitoring. 탄저병(Anthrax)은 탄저균(Bacillus anthracis) 감염에 의해 발생하는 인수공통전염병으로 감염경로에 따라 피부탄저, 장탄저, 폐탄저로 분류된다. 탄저균은 세 가지 독소(Protective antigen, Lethal factor, Edema factor)를 분비하며, 분비된 독소들은 순환계를 통해 전신으로 운반되어 각종 장기 및 혈관에서 출혈을 야기하고 숙주에게 치명적인 손상을 일으킨다. 특히 생물 무기로 악용될 수 있는 폐탄저의 경우 초기 증상이 감기와 유사하기 때문에 치료 시기를 놓칠 경우 치사율이 급격히 증가하게 된다. 따라서, 본 연구에서는 탄저균 감염을 빠르게 진단하고 탄저병 치료의 예후를 모니터링 할 수 있는 생물학적 표지인자(Biomarker)에 대한 연구를 수행하였다. 본 연구의 모델시스템인 인간 대동맥 내피세포(Primary human aortic endothelial cells, HAECs)에서 탄저 독소의 영향을 보기 위하여 cytotoxicity, cell growth inhibition 및 cell morphology를 관찰하였다. 그 결과 치사독소(Lethal toxin, LT)를 처리한 군이 대조군에 비해 농도 의존적으로 cell viability가 감소하는 것을 관찰할 수 있었으며, 이와는 대조적으로 부종독소(Edema toxin, ET)를 처리한 군에서는 세포의 morphology만 변할 뿐 cell viability는 대조군과 유사하게 유지되는 것을 확인할 수 있었다. 본 연구의 목적인 탄저병 biomarker 후보를 탐색하기 위하여 탄저 독소 LT와 ET에 의해 HAECs이 분비하는 단백질(Secretome)을 분리하여 nano-LC-ESI-MS/MS 기법으로 동정하였다. 그리고 후보단백질을 immunoblot 기법으로 verification하였으며, 그 결과 Lactotransferrin (LTF), Pregnancy zone protein (PZP), 그리고 Sarcolemmal membrane-associated protein (SLMAP)이 탄저병 biomarker 후보단백질로의 가능성이 있음을 확인하였다. 또한 탄저 독소 LT와 ET에 의해 HAECs에서 유리된 exosomal small RNA와 miRNA를 추출하여 각각 Expression array와 Microarray 기법을 통해 또 다른 탄저병 biomarker 후보를 동정하였다. 그 결과 exosomal small RNA 후보로는 ASPH, CD151, HSP90AA1, ICAM2, LYPD1, MPST, 그리고 NUAK1를 찾아내었고, exosomal miRNA 후보로는 hsa-miR-1307-3p를 찾아내었다. 그리고 후보 exosomal RNA를 qRT-PCR 기법을 통해 verification하였다. 더 나아가 탄저 독소를 주입한 rat으로부터 얻은 serum에서 본 연구에서 동정한 후보 biomarker에 대한 verification을 수행하여 clinical utility를 확인할 계획이다. 따라서, 본 연구에서는 탄저병의 세포배양 모델에서 탐색한 host-derived biomarker로서 단백질 3 종, mRNA 7 종, 그리고 miRNA 1 종을 동정 및 validation 하였으며, 이러한 11종의 proteomic 및 transcriptomic biomarkers가 탄저병의 진단과 치료 효과 모니터링에 사용될 가능성을 제시하였다.

Association between perceived exposure to secondhand smoke and depression independent of biomarker measured exposure

이동규 Graduate School, Yonsei University 2024 국내석사

서론 선행 연구들에서 간접흡연과 우울증의 상관성은 간접흡연의 정의 방식에 따라 서로 다른 결과들을 보인다. 간접흡연에 노출되었다고 인지한 정도와 우울증의 상관성은 유의한 반면, 바이오마커를 이용한 간접흡연 노출정도와 우울증의 상관성은 이보다 더 약하거나 유의하지 않은 결과들이 있다. 또한, 선행 연구들은 인지된 노출정도와 생물학적 노출정도를 동시에 고려하지 않았다. 이 연구는 성인 비흡연/과거흡연자들에서 인지된 간접흡연이 생물학적 간접흡연을 보정하고도 우울증과의 상관성이 있는지 연구하고자 한다. 연구방법 국민건강영양조사의 2014, 2016, 2018, 2020년 자료로부터 총 16,926명의 참가자들의 자료를 수집하였다. 해당 자료는 복합표본설계를 통해 인구집단 전체를 대표할 수 있도록 대상자를 추출한 자료이다. 독립변수인 인지된 간접흡연은 직장, 가정, 또는 공공장소 실내에서 최근 7일간 간접흡연에 노출된 경험이 있는지에 대한 이분형 변수로 정의되었다. 종속변수인 우울증은 한글판 우울증 선별도구 (Patient Health Questionnaire-9)에서 10점 이상인 경으로 정의되었다. 주요 공변수인 바이오마커 기반 간접흡연 노출정도는 니코틴 대사체인 코티닌을 이용하였다. 해당 연구는 로지스틱 회귀분석을 이용해 인지된 간접흡연과 우울증의 상관성이 바이오마커 기반 간접흡연 노출량을 보정하여도 통계적으로 유의한 양의 상관관계를 갖는지 알아보았다. 또한, 자살 (자살사고, 계획, 행동) 및 의료이용 (우울증 후 의료이용 및 정신과 질환으로 인한 의료이용) 관련된 변수들을 이차 종속변수로서 평가하였다. 추가로, 인지된 간접흡연 노출과 바이오마커 기반 간접흡연 노출의 우울증에 대한 교호작용에 대해 검증하였다. 그리고, 사회경제적 계층, 과거 흡연 여부, 그리고 성별에 따라 인지된 노출과 우울증의 상관성의 크기가 달라지는지에 대해 분석하였다. 그리고, 인지된 간접흡연 노출을 장소에 따라 나누어, 각각의 장소에서의 인지된 노출이 우울증과 상관성 있는지에 대해 연구하였다. 추가로, 여러 민감도 분석들을 실시하였다. 나이와 동거인수를 국민건강영양조사 데이터에서는 80세 이상은 80으로, 6명 이상은 6으로 코딩하기 때문에, 정보의 손실 가능성에 대해 나이와 동거인수를 범주형 변수로 변경해 분석하였다. 그리고, 단면연구로서의 연구설계의 한계점에 의한 역인과성의 가능성을 고려하기 위해 우울증에 대한 의사진단 경험이 없는 대상자들로 분석을 한정하여 시행하였다. 또한, 코티닌의 반감기가 짧아 만성적인 생물학적인 간접흡연 노출을 충분히 반영할 수 없어, 더욱 긴 반감기를 가지는 바이오마커를 이용해 동일한 분석을 수행하였다. 그 후, 현재 흡연자들 중 자가응답에서 비흡연/과거흡연자로 응답한 경우를 제외시키기 위해 바이오마커들을 이용하여 의심 대상자들을 재분류 한 후 분석을 수행하였다. 우울증에 대한 정의 또한 여러가지로 변경하여, 우울증 선별도구의 절단점을 변경하거나, 자기보고된 우울증상으로 종속변수를 변경하여 동일한 분석을 수행하였다. 마지막으로, 다중 대치법을 이용하여 분석을 재시행하였다. 또한, 종속변수를 변경하여 음성대조군 분석을 시행하였다. 인지된 간접흡연 및 바이오마커 기반 간접흡연 모두 통계적으로 유의하지 않은 상관성이 나오도록 백내장 및 B형 간염 병력을 비정신과 비호흡기 음성대조군 질환으로 선정하였다. 또한, 생물학적 간접흡연만 유의한 상관성이 보이도록 천식 병력을 비정신과 호흡기 음성대조군 질환으로 선정하였다. 결과 인지된 간접흡연은 생물학적 노출량을 보정하여도 우울증과 유의한 상관성을 보였다 (오즈비: 1.60, 95% 신뢰구간: 1.31-1.95). 또한, 인지된 노출은 자살사고 (오즈비: 1.34, 95% 신뢰구간: 1.11-1.61) 및 정신과 질환으로 인한 의료이용 (오즈비: 1.62, 95% 신뢰구간: 1.23-2.13)과도 상관성을 보였다. 인지된 노출과 바이오마커 기반 노출의 우울증에 대한 교호작용은 통계적으로 유의하지 않았다. 인지된 노출과 우울증의 상관성은 가장 고소득 집단 (오즈비: 3.31, 95% 신뢰구간: 2.03-5.39)에서 높았으며, 그 외의 소득계층에서는 상관성의 크기가 적었다. 예를 들어, 가장 저소득 집단이면서 인지된 간접흡연이 없을 때의 상관성 (오즈비: 3.86, 95% 신뢰구간: 2.60-5.75)은 가장 저소득 집단이면서 인지된 간접흡연이 있을때의 상관성 (오즈비: 4.99, 95% 신뢰구간: 3.24-7.69)와 차이가 적었다. 인지된 간접흡연 노출은 직장실내 (오즈비: 1.62, 95% 신뢰구간: 1.17-2.25), 가정실내 (오즈비: 1.56, 95% 신뢰구간: 1.14-2.13), 그리고 공공장소 실내 (오즈비: 1.57, 95% 신뢰구간: 1.28-1.93)에서의 노출 모두 우울증과 유의한 상관성을 보였다. 마지막으로, 여러 민감도 분석의 결과들은 기존 분석과 유사한 결과를 보였다 (오즈비 최솟값: 1.38, 최댓값: 1.71). 비정신과 비호흡기 음성대조군 질환은 인지된 간접흡연 (백내장과의 오즈비: 1.07, 95% 신뢰구간: 0.78-1.47, B형 간염과의 오즈비: 1.16, 95% 신뢰구간: 0.91-1.47) 및 바이오마커 기반 간접흡연 (백내장과의 오즈비: 0.95, 95% 신뢰구간: 0.86-1.05, B형 간염과의 오즈비: 1.00, 95% 신뢰구간: 0.94-1.06) 모두와 통계적으로 유의하지 않은 상관성을 보였다. 또한, 비정신과 호흡기 음성대조군 질환은 바이오마커 기반 간접흡연 (천식과의 오즈비: 1.09, 95% 신뢰구간: 1.01-1.18)만이 통계적으로 유의한 상관성을 보였다. 결론 인지된 간접흡연은 우울증에 대해 실제 생물학적인 노출량과 독립적으로 상관성을 보였다. 현재의 국민건강증진법은 생물학적인 간접흡연 노출량을 최소화시키는데 집중하고 있지만, 추가적으로 인지되는 간접흡연을 줄이기 위한 노력이 필요하다. 인구 집단을 간접흡연에 의한 우울증에서 보호하기 위해서는, 흡연 공간을 엄격하게 분리하거나 흡연실에서 불투명한 벽을 사용하는 것처럼 시각적 노출을 줄이거나, 환기를 시키거나 냄새가 덜 나는 형태의 담배를 사용하도록 하여 후각적 노출을 줄이는 등의 방식들이 필요할 수 있을 것이다. Introduction Studies on the association between secondhand smoke and depression show heterogeneous results according to the definition of exposure, whether assessed by perception or biomarkers of tobacco smoke. While associations between secondhand smoke and depression using definitions by perception showed significant results, associations were weaker or nonsignificant when associations between biomarker-based secondhand smoke exposure and depression was evaluated. Furthermore, no prior study evaluated both perceived and biomarker measured exposure to secondhand smoke simultaneously. This study aimed to evaluate whether perceived exposure was associated with depression after adjusting for biomarker measured exposure in an adult, non-/ex-smoker population. Materials and methods A total of 16,926 participants were sampled from the Korea National Health and Nutrition Examination Survey biennially from 2014 to 2020. The dataset followed a complex survey structure enabling representation of the general Korean population. Perceived exposure was defined as a binary variable indicating self-reported indoor secondhand smoke exposure in occupational, household, or public locations in the past 7 days. Log-transformed urine cotinine, a nicotine-specific metabolite, was used as the biomarker. Depression was defined using the Patient Health Questionnaire-9, where a total score of 10 or above indicated depression. Logistic regression accounting for the complex survey structure evaluated the association between perceived exposure and depression while adjusting for cotinine. In addition to the main analysis, variables associated with suicidality and medical use were analyzed as secondary outcomes of the study. Suicidal ideation, planning, and attempts were used as suicidality associated outcomes. Medical use after depression, and medical use due to any mental health problems were used as medical use outcomes. Also, interaction between perceived and biomarker measured exposure was assessed. Furthermore, effect heterogeneity across socioeconomic status, smoking history, and sex was evaluated. Next, associations between perceived secondhand smoke exposure in individual locations (i.e., occupational, household, or public locations) and depression were evaluated. Several sensitivity analyses were conducted to test the robustness of the results. First, age and the number of cohabitants were used as categorical variables instead of continuous variables. This was done in response to the lost information in the dataset, where participants with age over 80 or the number of cohabitants over 6 were coded as 80 and 6 respectively. To account for possible reverse causality due to the cross-sectional study design, the analysis was restricted to participants who were never diagnosed as depressed. Also, to account for the short half-life of cotinine, an alternative biomarker with a longer half-life was used for adjustment. Considering smokers who falsely report themselves as non-smoking, analysis was performed after excluding hidden smokers using urine biomarkers. Furthermore, different definitions of depression were used, such as changing the cut-off score of the questionnaire or using self-reported depressive symptoms. Finally, analysis was done after multiple imputation. Also, negative control analysis was done by changing the outcome variable. History of cataract and hepatitis B were used as non-psychiatric, non-respiratory outcomes, where both perceived and biomarker measured secondhand smoke exposure were expected to show nonsignificant associations with the outcome. Meanwhile, history of asthma was used as a non-psychiatric, respiratory negative control outcome, where only biomarker measured exposure was expected to show significant associations with the outcome. Results Perceived exposure was associated with depression (odds ratio [OR]: 1.60, 95% confidence interval [CI]: 1.31-1.95). Also, perceived exposure was associated with suicidal ideation (OR: 1.34, 95% CI: 1.11-1.61) and medical assistance due to psychiatric problems (OR: 1.62, 95% CI: 1.23-2.13). Interaction between perceived exposure and biomarker measured exposure on depression was insignificant. The perceived exposure-depression association was stronger in highest income groups (OR: 3.31, 95% CI: 2.03-5.39) while lower income groups showed lower increase in odds (e.g., lowest income without perceived exposure [OR: 3.86, 95% CI: 2.60-5.75], lowest income with perceived exposure [OR: 4.99, 95% CI: 3.24-7.69]), indicating negative additive interaction. Perceived exposure in occupational (OR: 1.62, 95% CI: 1.17-2.25), household (OR: 1.56, 95% CI: 1.14-2.13), and public (OR: 1.57, 95% CI: 1.28-1.93) locations were all associated with depression. Multiple sensitivity analyses reported results that were significant and similar to the main analysis, with ORs ranging from 1.38 to 1.71. Non-psychiatric, non-respiratory negative control outcomes showed nonsignificant associations with both perceived (OR:1.07, 95% CI: 0.78-1.47 for cataract, OR: 1.16, 95% CI: 0.91-1.47 for hepatitis B) and biomarker measured (OR: 0.95, 95% CI: 0.86-1.05 for cataract and OR: 1.00, 95% CI: 0.94-1.06 for hepatitis B) secondhand smoke exposure. Also, the non-psychiatric, respiratory negative control outcomes showed only significant associations with biomarker measured exposure (OR: 1.09, 95% CI: 1.01-1.18 for asthma). Discussion Perceived exposure to secondhand smoke was associated with depression independent of actual biological exposure. While the current National Health Promotion Act focuses on reducing biological tobacco smoke exposure, regulations focusing on restricting perceived exposure should be emphasized. Reducing perceivable cues such as visual (e.g., spatial separation, intransparent smoking room walls) and olfactory (e.g., ventilation, use of tobacco with less odor) cues may be further needed to protect the general population from depression.

Biomarker를 활용한 식품 중 다환방향족탄화수소류의 인체 위해성평가

윤은경 高麗大學校 2005 국내박사

This study was conducted to estimate excess cancer risk caused by current dietary polycyclic aromatic hydrocarbons (PAHs) exposure of the Korean and to identify the relationship between the internal and external doses of dietary intake using analysis of urinary 1-hydroxypyrene as a biomarker of PAHs. Sixty food commodities highly consumed containing high PAHs were selected from 2001 national health and nutrition survey. The foods including rice, bread, kimchi, soybean curd, ramen, chicken, pork etc. were analyzed for 14 PAHs congeners profile using high performance liquid chromatography and fluorescence detector. The range of total PAHs level was 8.31 ppb ~ 218.41 ppb and the highest total PAHs level was found in roasted laver (218.41 ug/kg). The higher levels of naphthalene and fluoranthene were detected in all foods except microwave cooked foods. Heating process of foods may result the rise in PAHs level and total PAH-TEQBaP level. Total PAHs contents detected in edible foods were significantly correlated with fluoranthene, benzo(a)anthracene, pyrene, and chrysene levels, having relative factor greater than 0.5. Considering the toxic equivalent (TEQ) level converted by the toxic equivalent factors (TEFs), the highest total PAH-TEQBaP level was detected from uncooked ramen because of its deep frying process. The highest total PAH-TEQBaP level of cooked foods was found in roasted laver as 1.2 ug-TEQ/kg. For the exposure assessment of PAHs from foods ingestion, the average body weight for each age groups was separated as 1-6yrs, 7-19yrs, 20-64yrs and >64yrs and consumed values from report on national health and nutrition survey were drived. The estimated lifetime average daily intake of PAHs was 3.2210^-3 ug/kg/day for carcinogenic effects and was higher in younger population than elder population. To calculate a realistic PAHs intake level, weekly intake frequency and one serving size were considered to calculate lifetime average daily intake of PAHs, which was 2.7910^-3 ug-TEQ/kg/day. The dietary excess cancer risk estimated using cancer potency of benzo(a)pyrene (7.3(mg/kg/day)^-1) was 2.3x10^-5 and 2.010^-5, for 3.2210^-3 ug-TEQ/kg/day and 2.7910^-3 ug-TEQ/kg/day respectively. This result implies the probability of tumor break-out in upper gastrointestinal tract was two per hundred thousand persons. For the identification of dose-response according pyrene exposure by food ingestion and urinary excretion of 1-hydroxypyrene, human monitoring was executed. Fifty-five non-smoking volunteers had three food items (fried chicken, hamburger, roasted pork) in every other day with three different amounts. In every the first morning, urine samples were collected during 18 consecutive days. Urinary concentration of 1-hydroxypyrene was measured by high performance liquid chromatography and fluorescence detector, and adjusted to urinary creatinine concentration. The results revealed that urinary 1-hydroxypyrene concentration was affected by diet intake in a numeric formula : y= 18.16x+0.102 (R=0.34, p<0.0001) according regression analysis. On the other hands, regression of the total PAHs-TEQ exposure level and pyrene exposure level induced numeric formula y= 1.334x+0.00003 (R=0.86, p<0.0001). It induced relative equation of dietary PAHs-TEQ exposure level and urinary 1-hydroxypyrene level, y(total PAHs-TEQ intake, ug-TEQ/kg/day) = 0.0076x (urinary 1-hydroxypyrene level, umol/ mol cr.) + 0.0068 when mixed the results of above two regression analyses. The calculation of excess cancer risk of the PAHs diatery exposure using biomarkers in human study has lower uncertinty level than the cancer risk calculated from food PAHs contamiantion level and shows more realistic PAHs exposure trends.

Study of Lung cancer Biomarkers:Identification and validation of two protein biomarkers from different sources : 혈액유래 단백질 바이오마커의 검증방법의 확립과 폐암조직 유래 단백질 바이오마커의 발굴과 그 기전연구

성혜진 경북대학교 대학원 2014 국내박사

Quantification is an essential step in biomarker development. Multiple reaction monitoring (MRM) is a new modified mass spectrometry-based quantification technology that does not require antibody development. Serum amyloid A (SAA) is a positive acute-phase protein identified as a lung cancer biomarker in our previous study. Acute SAA exists in two isoforms with highly similar (92%) amino acid sequences. Until now, studies of SAA have been unable to distinguish between SAA1 and SAA2. To overcome the unavailability of a SAA2-specific antibody, we developed MRM methodology for the verification of SAA1 and SAA2 in clinical crude serum samples from 99 healthy controls and 100 lung adenocarcinoma patients. Differential measurement of SAA1 and SAA2 was made possible for the first time with the developed isotype-specific MRM method. Most healthy control samples had small or no MS/MS peaks of the targeted peptides otherwise, higher peak areas with 10- to 34-fold increase over controls were detected in lung cancer samples. In addition, our SAA1 MRM data demonstrated good agreement with the SAA1 enzyme-linked immunosorbent assay (ELISA) data. Finally, successful quantification of SAA2 in crude serum by MRM, for the first time, shows that SAA2 can be a good biomarker for the detection of lung cancers. As lung cancer is showing the highest mortality in cancer related death, convenient and rapid molecular diagnostic methods might be helpful for the lung cancer diagnosis and its treatment. Protein biomarkers could be lead to the development of molecular diagnostics. Biomarkers from distal fluids generally have high cross reactivity to many similar diseases. To discover lung cancer tissue specific protein biomarkers, secreted proteins from primary cultured lung cancer and adjacent normal tissues from patients were subjected to proteomic analysis. QSOX1 (Quescin sulfhydryl oxidase), one of the proteins in the increased protein list, is selected as a biomarker candidate. Increase of QSOX1 in lung cancer was verified through western blot analysis of about 60 patients’ tissue lysates. Unique higher expression of QSOX1 in lung cancer compared to other solid cancers was confirmed by immunohistochemistry. Lewis lung adenocarcinoma mouse model experiment proved the QSOX1 increase in the induced lung cancer tissues of mice. QSOX1 is known as a secreted protein, therefore, QSOX1 level in the albumin and IgG depleted serum was measured by MRM (multiple reaction monitoring). Significant increase was detected in lung cancer patients compared to healthy control. In conclusion, QSOX1 might be a lung cancer tissue derived specific biomarker and in combination use with other blood biomarkers, it is expected to increase specificity of biomarkers in diagnostics.

Integrated assessment of multi-biomarker responses to hazardous chemicals in fish, Cyprinus carpio and Zacco platypus

Kim, Woo-Keun 고려대학교 대학원 2012 국내박사

Biomarker responses in fish have been widely used to assess toxicological effects of hazardous chemicals. Given that more than one biomarker response is generally observed by exposure to toxic compounds, the use of a battery of biomarkers is likely preferred and integration of the biomarker battery is one of the key challenges. Thus, this study aimed to evaluate biomarker responses in Cyprinus carpio (a common carp) and Zacco platypus (a pale chub) exposed to hazardous chemicals, and to integrate the multi-biomarkers quantitatively to provide a useful tool for ecotoxicological assessments. Firstly, the toxicological effects of perfluorooctanoic acid (PFOA) and perfluorooctanate (PFOS) toward C. carpio were evaluated by assessing the responses of five biomarkers, including DNA single-strand breaks (COMET), vitellogenin (VTG) concentration and the activities of 7-ethoxyresorufin-O-deethylase (EROD), acetylcholinesterase (AChE) and catalase (CAT). Upon PFOA exposure, both the VTG concentration and CAT activity were significantly increased, while there was a negligible change in the responses of other biomarkers when compared to the control. Upon PFOS exposure, a significant increase in the DNA single-strand breaks was observed, while the responses of other biomarkers were not significantly altered when compared to the control. Standardized scores of biomarker responses were computed as the integrated biomarker response (IBR) and visualized using star plots. PFOA and PFOS showed totally different patterns of biomarker responses, the IBR values were strongly correlated with the logarithmic concentrations of PFOA and PFOS (r2 = 0.943 and 0.951, respectively). Secondly, the toxicological effects of copper (Cu) and benzo[a]pyrene (BaP) toward Z. platypus were evaluated by assessing histological/physiological responses as well as the molecular/biochemical biomarkers. No significant differences at the histological or physiological levels were observed among treatment groups, except for kidney tissue exposed to 20 μg Cu L-1. However, various molecular/biochemical responses were observed in Z. platypus, and these primarily depended on exposure time. Upon Cu exposure, both COMET and metallothionein (MT) activity were significantly increased for 4 days, while there was no significant change after 14 days of exposure. BaP exposure significantly induced COMET and EROD activity at 4 and 14 days of exposure, however significant induction of AChE activity was observed only at 14 days of exposure. The IBR values well correlated with the concentrations of Cu and BaP, and the correlations were better at 4 days of exposure (r2 = 0.780 and 0.699, respectively) than 14 days (r2 = 0.692 and 0.548, respectively). Finally, the oxidative stresses of Cu and BaP toward Z. platypus were evaluated by assessing induction of antioxidant enzymes and lipid peroxidation in liver, muscle and gill of the fish. The most significant oxidative stresses were observed in liver among the target tissues. The 4 day-exposure to Cu resulted in increase of superoxide dismutase (SOD) activity in liver. BaP exposure, however, did not induce any antioxidant enzyme activities and lipid peroxidation, implying chemical specific antioxidant responses in Z. platypus. Additionally, the correlation (r2 = 0.780) between IBR value and Cu concentration was much improved by inclusion of the oxidative stress, SOD (r2 = 0.889).

Development of protein arrays for validating serological biomarkers and monitoring enzyme activities : 혈액 biomarker의 검증 및 효소 활성도 분석을 위한 단백질 칩 개발

Deok-Hoon Kong 강원대학교 대학원 2014 국내박사

마이크로 어레이 기술은 고속으로 대량의 시료를 분석할 수 있는 기술로써, 단백질체학과 혈청학적 진단에 필요한 기술 중에 하나이다. 따라서, 본 연구에서는 혈청 biomarker의 검증, 단백질 분해 효소의 활성도 분석 그리고 혈액 내 protein kinase A (PKA)의 활성도 분석을 위한 새로운 마이크로 어레이 기술의 개발에 관한 내용을 시사한다. 첫번째로, 항체 어레이 데이터 분석시 실험적인 오차를 최소화하기 위해 median-centering 방법과 TIS 분석에 의한 혈청 내 IgM 농도를 이용하여 median-centered/IgM-tagged-internal standard (TIS) assay 표준화 방법을 개발하였다. 먼저 정상 (n = 25)과 간경화 (n = 25), 간암 (n = 29) 그룹으로부터 얻은 혈청 샘플에서 6종류의 혈청 단백질을 분석하기 위하여 5종류의 표준화 방법을 평가하였다. 평가방법은 상관계수와 변동계수를 이용하여 평가하였다. 그 결과, IgM을 이용한 표준화 방법은 변동계수의 향상을 가져왔고, median-centered 표준화 방법은 상관계수의 향상에 효과가 있었다. 또한, 혈액 내 IgM을 분석하기 위해 분석법을 비교해본 결과, TIS assay는 ELISA 방법에 비해 효과적이고 경제적이며 재현성이 있다는 결과를 얻었다. 이를 바탕으로 median-centered/IgM-TIS assay 방법을 이용하여 6종류의 혈액 단백질을 분석한 항체 어레이 데이터를 표준화 하였고, 표준화된 fluorescence intensity의 distribution 분석법을 이용하여 혈액 biomarker의 평가와 ROC 분석법을 이용하여 간경화와 간암을 진단하였다. 진단결과, Apolipoprotein A-1은 간경화 biomarker, alpha-fectoprotein과 CRP 복합은 간암의 혈액 biomarker로써 가능성이 있음을 알 수 있었다. 따라서, median-centered/IgM-TIS assay 표준화 방법은 항체 어레이 데이터의 분석과 간질환 및 암 진단 혈액 biomarker의 평가를 위한 유용한 표준방법이다. 두번째로, 단백질 분해효소 활성도의 실시간 정량 분석을 위해 유리기질로부터 액체상으로의 위상전위를 이용한 펩타이드 어레이를 개발하였다. 펩타이드 어레이는 두 기능을 가진 cross-linker인 GMBS와 C-와 N-말단에 각각 cysteine과 TAMRA를 가진 펩타이드 기질을 이용하여 제작하였다. 위상전위를 이용한 펩타이드 어레이는 MMP-3에 의해 유리기질로부터 분해된 펩타이드의 분석을 통해 입증하였다. 본 연구는 성공적으로 MMP-3의 단백질 분해 활성도를 분석하였다. 또한, MMP-3 활성도에 의해 분해된 펩타이드의 농도 분석을 통해 Km, Vmax 그리고 IC50 상수를 도출하였다. 따라서, 본 연구에서 개발된 펩타이드 어레이는 단백질 분해 효소의 kinetics 정량 분석과 약물 개발을 위한 kinetics 정보 제공에 적절하다. 세번째로, PKA 활성도 분석을 위해 형광 인산화 센서를 이용한 펩타이드 어레이를 개발하였다. 본 연구에서는 혈액 내 PKA 활성도와 자가항체 농도를 분석하기 위해 PKA 기질인 kemptide와 cPKA 에레이를 사용하였다. PKA 활성도 분석 칩의 민감도는 Trion X-100에 의해 0.01 U/ml로 증가하였고 PKA 억제제인 PKI에 의해 PKA 활성도가 억제되었으며 0.5 ?M에서 최고의 억제효과를 볼 수 있었다. 또한, 정상 (n = 30)과 간암 (n = 30), 위암 (n = 30), 폐암 (n = 30) 그리고 대장암 (n = 30) 환자 그룹에서 얻은 혈청에서 sPKA 활성도와 자가항체를 분석한 결과, sPKA 활성도 분석에서 암 그룹에 대해 90%의 민감도와 90%의 특이도, 0.966 AUC 그리고 3.5 U/ml의 cutoff value를 얻을 수 있었다. 그러나, PKA 자가항체 농도 분석 결과, 정상과 암 그룹과의 차이를 볼 수 없었고, sPKA 활성도와 자가항체 농도가 상관관계가 없음을 알 수 있었다. 따라서, sPKA 활성도가 자가항체보다 암 진단을 위한 biomarker로써 가능성이 있음을 알 수 있었고, sPKA 활성도 분석 칩이 암 진단과 PKA에 관련된 질환 진단에 효과적인 분석법임을 시사하는 바이다. Microarray technology is a one of the key technology in proteomics and serodiagnosis because this technology is appropriate for the large-scale and high-throughput analysis. In this paper, we developed novel microarrays for validating serological biomarkers, monitoring proteolytic enzyme activity, and analyzing serological protein kinase A activity. First, we developed a novel median-centered/IgM-tagged-internal standard (TIS) assay normalization using median-centering and TIS assay-based determination of serum IgM concentrations for minimizing systemic experimental variation in the analysis of antibody array data. We evaluated five normalization methods by analyzing correlation coefficients and coefficients of variation for six serum proteins using human serum samples from normal controls (n = 25) and patients with liver cirrhosis (n = 25) or hepatocellular carcinoma (HCC; n = 29). Median-centered normalization improved correlation coefficients, while IgM-based normalizations improved coefficients of variation. The TIS assay was more efficient, economical, and reproducible for determining IgM concentrations than enzyme-linked immunosorbent assay. Additionally, we normalized antibody array data for six serum proteins using the median-centered/IgM-TIS assay, and evaluated serum biomarkers through distribution analysis of normalized fluorescence intensities and receiver operating characteristic analyses for the diagnosis of liver cirrhosis and HCC. Apolipoprotein A-1 and a combination of alpha-fetoprotein and C-reactive protein were determined to be potential serological biomarkers for liver cirrhosis and HCC, respectively. Thus, median-centered/IgM-TIS assay normalization is a useful approach for analyzing antibody array data and evaluating serological biomarkers for the diagnosis of liver disease or cancers. Second, we have developed an assay using peptide arrays based on phase transition from the glass substrate to the liquid for monitoring quantitative protease activity in real-time. Peptide arrays were fabricated using a bifunctional cross-linker, N-[γ-maleimidobutyryloxy] sulfosuccinimide ester, and a substrate peptide containing two functional groups, cysteine and tetramethyl-6-carboxyrhodamine (TAMRA) on the C- and N-terminus, respectively. The phase transition-based peptide arrays were characterized by analyzing the substrate peptide cleaved from the solid substrate by matrix metalloproteinase-3 (MMP-3). We successfully used this assay to determine quantitative proteolytic activity of MMP-3 in a dose-dependent manner. In addition, parameters including Michaelis constant (Km), maximum rate of enzymatic reaction (Vmax), and half maximal inhibitory concentration (IC50) were determined by analyzing the concentrations of substrate peptide cleaved by MMP-3. Therefore, this new assay has potential for the quantitative analysis of enzyme kinetics of protease and informs research developments in drug discovery utilizing kinetic studies. Third, we present a novel peptide array based on fluorescence phosphorylation sensor for analysis of c-AMP dependent protein kinase (PKA) activity. In the present study, we used kemptide (the protein kinase A [PKA] substrate peptide) and PKA catalytic subunit C? (cPKA) arrays to evaluate serological PKA (sPKA) activity and autoantibody levels, respectively. The sensitivity of the on-chip sPKA activity assay was enhanced by Triton X-100, with a 0.01 U/mL detection limit. Protein kinase peptide inhibitor (PKI) decreased PKA activity in a dose-dependent manner, with a maximal effect at 0.5 ?M. We analyzed sPKA activities and autoantibody levels in sera from normal individuals (n = 30) and patients with hepatic (n = 30), gastric (n = 30), lung (n = 30), or colorectal (n = 30) cancers. sPKA activities in human sera from cancer patients were much higher than those in controls. The sPKA activity assay for cancer patients (n = 120) showed 90.0% sensitivity and 90.0% specificity at the cutoff value of 3.5 U/mL, with an area under the curve value of 0.966. However, no significant differences in PKA autoantibody levels were found between groups. Additionally, sPKA activities were not correlated with sPKA autoantibody levels. These results suggest that sPKA activity rather than autoantibody level could be used as a biomarker for cancer diagnosis. Furthermore, on-chip sPKA activity assay could be useful for cancer diagnosis and investigating PKA-related human diseases.

Evaluating the Utility of S100A8/A9 as a Non-Invasive Biomarker

Mellett, Leah Washington University in St. Louis ProQuest Disser 2023 해외박사(DDOD)

Biomarkers, a portmanteau of "biological markers", are clinically relevant observations that can accurately and reproducibly indicate or predict patient outcomes. These objective measurements have vast clinical utility as they can aid clinicians with diagnoses, provide information about patient prognoses, and monitor changes in patient health statuses. While the discovery of several biomarkers are attributed to differential expression or concentration of genes or proteins within blood samples, biomarkers can be alternatively found in other bodily fluids or tissues depending on the associated condition. Recently, advances in urine proteomics have allowed for the identification of urine biomarkers that may eliminate the drawbacks associated with serum biomarkers. Identifying and evaluating urinary biomarkers has the potential to significantly increase patient comfort and accessibility to proper healthcare. To this end, our lab had previously investigated calprotectin (S100A8/A9) as a potential biomarker for tuberculosis (TB) as elevated serum concentrations were observed in patients with active forms of the disease. S100A8/A9 is a calcium-binding alarmin that mediates proinflammatory responses by binding to toll-like receptor 4 (TLR4) and the receptor for advanced glycation end products (RAGE). Comprising approximately 45% of the cytoplasmic proteins present in human neutrophils, it is a sensitive marker for neutrophil activation and has been associated with several inflammatory diseases. Severe COVID-19 cases are often characterized by cytokine storm, which is a dysregulated immune response involving the release of over 150 inflammatory cytokines. Studies published in the early period of the COVID-19 pandemic suggested that S100A8/A9 expression may be associated with cytokine storm and the formation of neutrophil subsets that were observed only in severe COVID-19 cases. Similar to our findings from initial TB studies, several non-US studies reported that S100A8/A9 serum levels were significantly elevated in patients with severe and fatal COVID-19 cases as compared to patients with mild forms of the disease. Because S100A8/A9 levels have been quantified in both serum and urine, we hypothesized that the trends observed in serum were mirrored in urine samples.In pursuit of evaluation of urine S100A8/A9 as a biomarker for COVID-19, I used protein quantification to demonstrate that urine S100A8/A9 cannot accurately indicate COVID-19 patient outcomes. In contrast, we found that serum S100A8/A9 levels mimicked results from non-US cohorts in their ability to distinguish COVID-19 disease severity. The lack of correlation between serum and urine S100A8/A9 levels suggested that previous findings of correlated S100A8/A9 values in patients with other conditions were likely disease-dependent.While the evaluation of urine S100A8/A9 as a marker for COVID-19 was unfavorable, we further delineated the utility of urine S100A8/A9 in the context of urinary tract infections (UTIs). While other studies accounted for patient UTI diagnosis as a confounding factor when determining the biomarker capacity of urine S100A8/A9 for kidney-related conditions, the utility of urine S100A8/A9 in diagnosing human UTIs was unexplored. We discovered that urine S100A8/A9 levels could predict both UTI diagnosis and clinically significant urine culture growth with a higher sensitivity than canonical markers for UTI, such as leukocyte esterase levels and white blood cell (WBC) presence. Collectively, this work highlights that S100A8/A9 is a promising target for the development of future, non-invasive diagnostic tools. In an attempt to address the challenges associated with the use of serum biomarkers, I have contributed to our understanding of the indicative and predictive capacity of S100A8/A9 in both serum and urine samples, and I have detailed a new potential utilization for urine S100A8/A9 in the context of urinary tract infections, which was previously unexplored.

Biomarker Development of Distant Metastatic Breast Cancer and Mood Disorders using Quantitative Proteomics and Bioinformatics

신동윤 서울대학교 대학원 2022 국내박사

서론: 액체 크로마토그래피 및 질량 분석법 기반 단백체 접근법이 특정 질병 및 장애와 관련된 바이오 마커를 발굴하고 개발하기 위해 적용되었다. 액체 크로마토그래피 고해상도 질량 분석법을 기반으로하는 비표적 단백체학은 수천 개의 단백질의 식별과 정량을 동시에 가능하게 하여 소량의 샘플에서 수백 개의 차등 발현 단백질을 생성한다. 액체 크로마토그래피 다중반응겁지 질량 분석법을 포함한 표적 단백체학은 높은 민감도, 정확도 및 재현성을 기반으로 표적 단백질을 정량하는데 사용된다. 임상 단백체학 연구에서 포르말린 고정 파라핀 포매조직절편 (FFPE), 혈액 및 기타 체액과 같은 임상 코호트에서 수집된 병리 및 임상 검체가 분석되었다. 임상 단백체학 분석의 경우 액체 크로마토그래피 및 질량 분석법 기반 접근법은 생체표지자의 발굴 및 개발과 높은 처리량과 높은 민감도로 임상 진단에 기여하는 강력한 기술이다. 또한, 액체 크로마토그래피 및 질량 분석법에 기반한 단백체학 연구는 특정 질병과 장애의 생물학적 및 분자적 특징에 대한 이해에 기여할 것이다. 방법: 1장에서는 필터 보조 검체 준비 (FASP), 연속 질량 꼬리 표지, 높은 산도 분획 및 액체 크로마토그래피-고분해능-질량분석법을 결합하여 원격 전이성 유방암 및 비원격 전이성 유방암의 포르말린 고정 파라핀 포매조직절편(FFPE)을 사용하여 심층 단백질 프로파일링 데이터를 획득하기 위한 통합 비표적 단백질 접근법이 적용되었다. 통계 분석은 차등 발현 단백질을 결정하고 원격 전이성 유방암을 예측하기 위한 후보 생체표지자를 발굴하기 위해 수행되었다. 원격 전이성 유방암의 분자 특성을 조사하기 위해 차등 발현 단백질 사용하여 유전자 온톨로지, 질병 및 기능, 표준 경로와 관련하여 생물정보학 분석이 수행되었다. 또한 후보 생체표지자의 원격 전이 가능성을 검증하기 위해 실시간 중합효소 연쇄 반응과 침입/이주 분석을 수행했다. 제2장에서는 크로마토그래피-다중반응검지-질량분석법에 기반한 표적 단백질 접근 방식을 적용하여 임상 코호트의 혈장 검체 에서 주요 우울 장애 및 양극성 장애와 관련된 단백질 후보 생체표지자를 정량 했다. 기술적 편차를 줄이기 위해 크로마토그래피-다중반응검지-질량분석법 데이터의 배치 효과 보정이 수행되었다. 이후 발현 양에 차이를 보이는 후보 단백질 생체표지자를 결정하기 위해 통계분석이 수행되었고, 특징 추출, 교차 검증 및 가중 모델 평균화를 결합한 최소 절대 수축 및 선택 연산자에 기반한 머신 러닝 접근법이 주요 우울 장애 와 양극성 장애를 구별하기 위한 잠재적 진단 모델을 개발하기 위해 수행되었다. 또한, 모델에 포함된 단백질과 기분 장애 사이의 생물학적 관계를 조사하기 위해 생물정보학 기반 네트워크 분석을 수행하였다. 결과: 1장에서 포르말린 고정 파라핀 포매조직절편-연속 질량 꼬리 표지 풀링 샘플 세트와 포르말린 고정 파라핀 포매조직절편-연속 질량 꼬리 표지 개별 샘플 세트로부터 각각 원격 전이 및 비원격 전이 그룹을 비교한 총 9,441개 및 8,746개의 단백질이 동정 되었다. 또한, 저침습성 및 고침습성 세포주를 비교한 유방암 세포주-연속 질량 꼬리 표지 샘플 세트에서 총 7,823개의 단백질이 동정 되었다. 후보 생체표지자의 단계별 결정 기준에 따라 2개의 단백질(LTF, TUBB2A)을 유방암 원격전이 예측을 위한 후보 생체표지자로 결정하였다. 14개 유방암 세포주의 RT-PCR 데이터의 LTF와 TUBB2A 발현 패턴을 크로마토그래피-질량분석 데이터의 발현 패턴과 비교했을 때, TUBB2A만이 두 데이터 사이에서 일관된 발현 패턴을 보였다. 그 결과, TUBB2A는 이후 원격 전이 활성이 검증되는 새로운 생체표지자 후보로 선정되었다. 또한 생물정보학적 결과를 통해 원격 전이의 전반적인 분자적 특징을 도출하였으며, 유방암 아형 간 원격 전이성 유방암의 분자 기능 차이를 입증하였다. 제2장에서는 270명의 혈장 샘플[90명의 주요 우울 장애, 90명의 양극성 장애, 90명의 정상 대조군]에서 주요 우울 장애 및 양극성 장애 에 관한 671 펩타이드에 해당하는 총 210개의 단백질표적을 크로마토그래피-다중반응검지-질량분석법을 사용하여 안정적으로 정량 하였다. 훈련 세트(72명의 주요 우울 장애 및 72명의 양극성 장애)에서는 9개의 혈장 단백질로 구성된 일반화 가능한 모델이 개발되었다. 모델은 테스트 세트(18명의 주요 우울 장애 및 18명의 양극성 장애)에서 평가되었다. 이 모델은 훈련 (곡선 아래의 면적 = 0.84)과 테스트 세트(곡선 아래의 면적 = 0.81)에서 MDD를 BD와 구별하고 현재 고조증/저조증/혼합 증상 (90명의 주요 우울 장애 및 75명의 양극성 장애)(곡선 아래의 면적 = 0.83)에서 우수한 성능(곡선 아래의 면적 > 0.8)을 보였다. 그 후, 이 모델은 약물 투여 경험이 없는 주요 우울 장애와 양극성 장애 환자 (11명의 주요 우울 장애 및 10명의 양극성 장애)(곡선 아래의 면적 = 0.96)에서 우수한 성능을 보였고, 주요 우울 장애 대 정상 대조군(곡선 아래의 면적 = 0.87) 및 양극성 장애 대 정상 대조군 (곡선 아래의 면적 = 0.86)에서 우수한 성능을 보였다. 또한, 9개의 단백질은 신경, 산화/질소 스트레스, 면역/염증 관련 생물학적 기능과 관련이 있었다. 결론: 제1장에서, 본 연구는 포르말린 고정 파라핀 포매조직절편 조직을 사용하여 가장 큰 원격 전이성 유방암 단백체를 처음으로 구축하였다. 깊이 있는 단백체 데이터를 통해 새로운 생체표지자 후보와 원격 전이성 유방암의 단백체 특성을 발견할 수 있었다. 다양한 유방암 아형에서 원격 전이성 유방암의 뚜렷한 분자적 특징도 확립되었다. 우리의 단백체 데이터는 원격 전이성 유방암 연구에 귀중한 자원을 제공한다. 제2장에서는 표적 단백체학 접근방식을 사용하여 주요 우울 장애 및 양극성 장애 환자를 구별 가능성을 제안했다. 우리는 주요 우울 장애와 양극성 장애를 구별하기 위해 9개 혈장 단백질로 구성된 일반화 가능한 모델을 개발했다. 우리의 결과는 이러한 장애가 개발된 모델을 사용하여 구별 및 진단 할 수 있음을 시사한다. 또한, 우리는 9개의 혈장 단백질이 우울 장애와 양극성 장애와 생물학적으로 중요한 연관성을 가질 것을 제안한다. Introduction: Liquid chromatography (LC)-mass spectrometry (MS)-based proteomic approaches have been applied to discover and develop biomarkers that are associated with specific diseases and disorders. Untargeted proteomics based on LC-high resolution MS has enabled simultaneous identification and quantification of thousands of proteins and hundreds of differentially expressed proteins (DEPs) in small amounts of samples. Targeted proteomics including LC-multiple reaction monitoring (MRM)-MS has been used to quantify interesting proteins with high sensitivity, accuracy, and reproducibility. Numerous clinical proteomics studies employ pathological and clinical specimens collected from clinical cohorts such as formalin-fixed paraffin-embedded (FFPE) tissues, blood, and other body fluids, . For clinical proteomic analysis, LC-MS-based approaches are powerful technologies in discovery and development of biomarkers with their high throughput and high sensitivity. In addition, proteomic studies based on LC-MS will contribute to understanding of biological and molecular features of specific diseases and disorders. Methods: In chapter I, an integrated untargeted proteomic approach that combined filter-aided sample preparation (FASP), tandem mass tag labeling (TMT), high pH fractionation, and LC-high resolution-MS was applied to acquire in-depth proteomic profiling data from FFPE tissues of distant metastatic breast cancer patients collected from a clinical cohort. Statistical analyses were performed to determine DEPs and discover candidate biomarkers for predicting distant metastatic breast cancer. Bioinformatics analyses were performed to examine molecular characteristics of distant metastatic breast cancer. In addition, in vitro assays were performed to validate distant metastatic potential of candidate biomarkers. In chapter II, targeted proteomic approach based on LC-MRM-MS was applied to quantify protein targets associated with major depressive disorder (MDD) and bipolar disorder (BD) in plasma samples collected from a clinical cohort. Batch-effect correction of LC-MRM-MS data was performed to reduce technical variations. Subsequently, univariate analysis was performed to determine proteomic candidate features, and machine learning approaches were performed to develop a potential diagnostic model for discriminating MDD and BD. In addition, network analysis was performed to examine biological associations between proteins included in the model and mood disorders. Results: In chapter I, a total of 9,441 and 8,746 proteins were identified from FFPE-TMT pooled samples set and FFPE-TMT individual samples set comparing distant metastasis and non-distant metastasis groups, respectively. In addition, 7,823 proteins were identified from the TMT-labeled breast cancer cell lines set comparing low invasive and high invasive cell lines. Two proteins (LTF and TUBB2A) were determined as candidate biomarkers. As a result, TUBB2A, which maintained consistent expression patterns between different quantitation platforms, was selected as a novel biomarker candidate. TUBB2A showed potential of distant metastatic activities. In addition, distinct alterations of proteome and molecular functions of distant metastatic breast cancer between breast cancer subtypes were demonstrated. In chapter II, 210 protein targets corresponding to 671 peptides pertinent to MDD and BD were stably and reproducibly quantified by LC-MRM-MS in individual plasma samples. In the training set, nine plasma protein biomarkers were developed and a generalizable model comprised of the nine proteins was constructed. The model demonstrated good performance (AUC > 0.8) in discriminating MDD from BD in the training (AUC = 0.84) and test sets (AUC = 0.81) and in distinguishing MDD from BD without current hypomanic/manic/mixed symptoms (AUC > 0.83). Subsequently, the model demonstrated excellent performance for drug-free MDD vs BD (AUC > 0.96) and good performance for MDD vs HC (AUC > 0.87) and BD vs HC (AUC > 0.86). Furthermore, the nine proteins were associated with neuro, oxidative and nitrosative stress, and immunity and inflammation-related biological functions. Conclusions: In chapter I, I constructed the largest FFPE tissue proteome of distant metastatic breast cancer proteome using. The depth of our dataset allowed us to discover a novel biomarker candidate as well as the proteomic characteristics of distant metastatic breast cancer. Distinct molecular features of various breast cancer subtypes were also established. Thus, our proteomic data can serve as a valuable resource for research on distant metastatic breast cancer. In chapter II, the viability of discriminating MDD and BD patients using a targeted proteomic approach was proposed. Our results suggest that the nine plasma proteins and their combined model has the potential to discriminate between MDD and BD patients and help diagnostic decision-making. Through both studies, the potential of LC-MS-based proteomics in the discovery and development of biomarkers was demonstrated.

Serum biomarker discovery related to pathogenesis in acute coronary syndrome by proteomic approach

Shin, Mi ji 을지대학교 대학원 2021 국내석사

Acute coronary syndrome (ACS) is a disease with extremely high morbidity and mortality worldwide, which is an ischemic symptom that develops due to the inadequate supply of blood flow from the coronary arteries to the heart. The biomarkers currently in use are biomarkers of necrosis that increase after myocardial necrosis, and there are limitations because these biomarkers do not show significant levels for diagnosis immediately after symptoms such as chest pain and can also increase in other diseases inducing myocardial necrosis. For these reasons, we aimed to discover new ACS diagnostic biomarkers with high sensitivity and specificity and related to pathogeneses of plaque rupture inducing ACS particularly. A discovery set consisted of 50 ACS patients and 50 healthy controls and analyzed their sera using mass spectrometer to identify proteins that were significantly different in each group. After selecting statistically significant protein candidates among these proteins, a validation set consisted of 120 people in each group to verify whether the protein candidates were suitable for biomarker. As a result, α-1-acid glycoprotein 1, complement C5, leucine-rich α-2-glycoprotein and vitronectin were found as biomarkers that increase in ACS patients, and gelsolin as biomarkers that decrease in ACS patients. These biomarkers have been shown to be associated with the pathogeneses of ACS and can predict the onset of ACS prior to the necrotic biomarkers currently in use. 급성 관상동맥 증후군은 관상 동맥으로부터 심장으로 흐르는 혈액 공급이 원활하지 않기 때문에 발병하는 허혈성 임장 증상을 말하는 것으로, 전 세계적으로 이환율 및 사망률이 매우 높은 질병이다. 현재 사용되는 바이오마커는 심근이 괴사된 후부터 증가하는 괴사 바이오마커로, 흉통과 같은 증상 직후 진단에 유의한 값을 나타내지 않으며, 급성 관상동맥 증후군이 아닌 심근에 괴사가 유도되는 다른 질병에서도 증가하기 때문에 한계점이 있다. 이와 같은 이유로 급성 관상동맥 증후군을 진단하기 위한 민감도와 특이도가 높은 새로운 진단 바이오마커를 발굴하는 것을 목적으로 본 연구를 진행하였으며, 특히 급성 관상동맥 증후군을 유도하는 플라크 파열의 발병 기전과 연관된 진단 바이오마커를 발굴하고자 하였다. Discovery set은 급성 관상동맥 증후군 환자군과 정상군을 각 50명씩으로 구성하여 혈청을 질량분석기를 통해 분석하였으며, 각 군에서 유의하게 차이나는 단백질들을 동정하였다. 해당 단백질들 중, 통계적으로 유의한 단백질 후보군들을 선별한 뒤, validation set에서 각 군당 120명씩으로 구성하여 해당 단백질 후보군들이 바이오마커로서 적합한지를 검증하였다. 그 결과, α-1-acid glycoprotein 1, complement C5, leucine-rich α-2-glycoprotein, vitronectin은 급성 관상동맥 증후군 환자군에서 증가하는 바이오마커로서, gelsolin은 감소하는 바이오마커로서 발굴되었다. 이들 바이오마커는 급성 관상동맥 증후군의 발병 기전과 관련이 있는 것으로 나타났으며, 현재 사용하는 괴사 바이오마커보다 더 이전에 급성 관상동맥 증후군의 발병을 예측할 수 있는 조기 진단 바이오마커로서의 사용이 가능할 것이다.

Diagnostic biomarkers for malignant pleural mesothelioma

김민규 Graduate School, Yonsei University 2022 국내박사

Malignant pleural mesothelioma (MPM) is a rare, aggressive cancer being developed on outlayer of lung tissues and caused by mostly occupational exposure to asbestos. Poor prognosis needs to develop the therapeutic drug as well as early diagnostic biomarker. Currently, mesothelin (MSLN), fibulin-3 (EFEMP1) and calretinin (CALB2) have been reported as potential diagnostic biomarkers for MPM. In this study, I first performed bioinformatics analysis for public database to find diagnostic biomarkers for MPM. From the analysis using Cancer Cell Line Encyclopedia (CCLE) and Gene Expression Omnibus (GEO) databases, included were 7 genes involving LOX, LOXL1, LOXL2, ZFPM2, THBS2, SULF1, CDH11 identified as potential diagnostic biomarkers. Among them, LOX family and ZFPM2 have not been reported related to MPM. Biostatistical analysis of receiver operating characteristic (ROC) analysis using the GEO database revealed similar diagnostic potential of LOX family and ZFPM2 genes compared to the known biomarkers involved EFEMP1, one of the best diagnostic biomarkers currently reported. Further molecular approach using quantitative real-time polymerase chain reaction (QPCR) confirmed the higher mRNA expression of these candidates in MPM cell lines and patient samples. Moreover, LOX showed MPM specific patterns of mRNA expression that were further confirmed in protein level by western blot assay. From this study, these novel biomarkers combined with the known MPM biomarkers would be used as multi-biomarker panels for MPM diagnosis. 악성흉막중피종은 각종 장기를 덮는 단층편평상피인 중피라 불리는 조직 중에 폐를 덮는 흉막에서 발병하는 희귀한 질병으로서 악성도가 매우 높고 효과적인 치료법이 아직 존재하지 않기 때문에 환자 예후가 매우 나쁜 것으로 알려져 있다. 악성흉막중피종은 과거에 각종 산업 환경에서 유용하게 사용되었던 석면에 노출되었을 때 발병하는 것이 확실하게 알려져 있으며, 긴 잠복기로 인하여 발병률은 점차 증가하는 추세이다. 이러한 특징들로 인하여 조기 진단력을 높이기 위한 진단용 바이오마커의 개발이 필요하다. 최근에 Mesothelin (MSLN), Fibulin3 (EFEMP1), Calretinin (CALB2) 등이 활용될 수 있는 진단 바이오마커로서 알려져 있다. 본 연구에서는 새로운 악성흉막중피종의 바이오마커를 발굴하기 위해 생물정보학적인 접근을 통해 공개데이터베이스 분석을 실시하였다. 두가지 공개 데이터베이스들(Cancer Cell Line Encyclopedia, Gene Expression Ominibus)을 이 용하여 ‘LOX, LOXL1, LOXL2, ZFPM2, THBS2, SULF1, CDH11’ 7개의 유전자들을 잠재적인 악성흉막중피종의 바이오마커로서 발굴하였으며, 이들 중 LOX, LOXL1, LOXL2, ZFPM2. 이렇게 4개의 유전자들은 아직까지 악성흉막중피종과 의 연관성이 밝혀진 바가 없다. 통계적 분석(ROC curve, receiver operating characteristic)을 통해 선택된 4개의 유전자들과 이미 알려진 바이오마커들 (EFEMP1, MSLN, CALB2)과 진단력을 비교하였을 때 비슷하거나 우월한 진단력 을 보이는 것으로 확인하였다. 또한 악성흉막중피종의 환자조직샘플을 확보하여, 발굴된 유전자들의 mRNA와 단백질 발현량을 일반환자조직샘플과 비교하였다. 이미 알려진 바이오마커들과 더불어 발굴된 후보유전자들 모두 악성흉막중피종 환자 샘플에서 발현량이 높은 것을 확인하였다. 따라서, 이미 알려진 바이오마커들과 본 연구에서 발굴한 새로운 유전자들을 함께 진단용 다중 바이오마커 패널로서 포함한다면 악성흉막중피종의 진단력을 개선시킬 수 있을 것으로 기대한다.