Protein Significance Analysis of Multiple Reaction Monitoring (MRM) Data via Generalized Linear Mixed Effect Models

학위논문 (석사)-- 서울대학교 대학원 : 통계학과, 2016. 2. 박태성.단백질 바이오마커의 발굴은 현재 생물의학 연구의 중요한 쟁점 중 하나이다. 엘라이자(enzyme-linked immunosorbent assay, ELISA) 는 전통적인 단백질 정량 방법의 하나다. 많은 수의 새로운 단백질들이 연구됨에 따라 엘라이자를 이용한 단백질 바이오마커 발굴에 있어 새로운 쟁점들이 드러났다. 다중반응관측(multiple reaction monitoring, MRM) 질량분석 방법은 엘라이자를 대체할 수 있는 특정 단백질 정량 방법이며 최근 더욱 활용되고 있다. 그러나 이러한 다중반응관측 자료를 이용한 통계적인 단백질 연관성 분석 방법의 개발은 크게 주목받지 못하였다. 초기에는 다중반응관측 자료를 이용하여 두 집단의 평균에 차이를 검증하기 위해 t 검정 혹은 쌍체 t 검정이 이루어 졌고 여러 집단의 평균 차이를 검증하기 위해 선형모형을 이용한 방법이 적용되었다. 2012년에 MSstats 이라 불리는 선형혼합모형을 이용한 단백질 연관성 분석 방법이 제안되었고 이후 널리 사용되고 있다. 이 선형혼합모형을 이용한 방법은 Skyline 프로그램과 R 프로그래밍 언어를 통해서 사용할 수 있다. 선형혼합모형을 통해 계산된 단백질 연관성 p 값은 모형 설정에 따라 많은 변화가 있고 이로 말미암아 많은 거짓 양성 혹은 참 음성이 생길 수 있다. 더욱이 이전에 제안된 선형혼합모형을 이용한 방법은 단백질을 대표하는 특징요인들의 집단 간 발현 양상이 서로 다르게 되면 검정력에 손실이 생기게 된다. 이러한 요인들이 임의효과인지 고정효과인지 에 대해 더욱 강건하며 특징요인들의 집단 간 발현 양상에도 더욱 강건한 모형을 제안하는 동기가 되었다. 우리는 일반화 선형혼합모형의 분산성분 검정방법을 이용한 단백질 연관성 분석을 제안하였다. 우리는 일반화 선형혼합모형 방법이 이전에 제안되었던 선형혼합모형 방법보다 요인들의 효과의 유형에 대해 더욱 강건하다는 것을 다양한 모의실험을 통해 관찰하였고 더욱 검정력이 좋은 경우를 다양한 모의실험을 통해 관찰하였다. 그뿐만 아니라 특징요인들의 집단 간 발현 양상이 서로 다를 때에 선형혼합모형 방법은 저조한 검정력을 보이지만 새롭게 제안한 일반화 선형혼합모형을 이용한 방법은 검정력에 손실이 없었다. 새롭게 제안한 일반화 선형혼합모형 방법이 유의미한 p 값을 제시하고 이전에 제안되었던 선형혼합모형 방법이 유의미하지 않은 p 값을 제시하는 경우를 실제 자료 분석을 통해 관찰하였다. 따라서 다중반응관측 자료를 이용해 단백질 연관성 분석을 할 때에는 이전에 제안되었던 선형혼합모형을 이용한 방법뿐만 아니라 새롭게 제안한 일반화 선형혼합모형을 이용한 방법도 같이 사용되어야 한다.Discovering protein biomarkers is one of the current important issues in biomedical research. The enzyme-linked immunosorbent assay (ELISA) is one of traditional protein quantitation techniques. As many novel proteins being studied, some issues of using ELISA for protein biomarker were emerged. The multiple reaction monitoring (MRM) mass spectrometry is a method for targeted protein quantification as well as an alternative of ELISA and has been widely utilized recently. However, development of statistical methods for this MRM data was not significant. In early analysis for MRM data, basic statistical methods such as two sample or paired t-test and linear models were employed. In 2012, statistical methods for protein significance analysis using linear mixed model (LMM) called MSstats was proposed and it has been widely used since then. This LMM approach is implemented on Skyline program and R programming language. The resultant protein significant p-value form this LMM approach is diversified for same data set depending on the model setting which could provide many false positives and many true negatives. Furthermore, there is a loss of power of this previously proposed LMM approach if some features behave oppositely from the others. These characteristics motivated us to develop a model that is robust on the true effect type and the behaviour of features among the groups so that the model provides robust p-values for protein significance analysis. We proposed variance component test of generalized linear mixed model (GLMM) approach for protein significance analysis. Through various simulation studies, we observed that the proposed GLMM approach is more robust on the type of effect and more powerful when there is the interaction effect between features and groups. Moreover, there is no loss of power of proposed GLMM approach when there are oppositely behaving features while LMM approach performed poorly. In real data analysis, we observed cases that the previously proposed LMM approach hardly detects while the GLMM approach provided significant p-values. Consequently, not only previously proposed LMM approach but proposed GLMM approach should be considered for protein significance analysis using MRM data.1. Introduction 7 1.1 Background 7 1.2 Purpose 8 2. Materials and Methods 9 2.1 Experimental Design of the Multiple Reaction Monitoring Mass Spectrometry 9 2.2 Linear Mixed Model (LMM) Approach 10 2.3 Generalized Linear Mixed Model (GLMM) Approach 11 3. Simulations 13 3.1 Simulation Settings 13 3.1.1 Settings for Type I Error Estimation 14 3.1.2 Settings for Empirical Power Comparison 14 3.1.3 Settings for Prediction Performance Comparison 15 3.2 Results of Simulation 16 3.2.1 Results for Type 1 Error Estimation 16 3.2.2 Results for Empirical Power Comparison 21 3.1.3 Results for Prediction Performance Comparison 26 4. Application to Real Data 29 4.1 Sorafenib Drug Response MRM Data 29 4.2 Results 30 5. Discussions 34 Bibliography 36 Abstract (Korean) 39Maste

Two Novel Bacteriophages Improve Survival in Galleria mellonella Infection and Mouse Acute Pneumonia Models Infected with Extensively Drug-Resistant Pseudomonas aeruginosa

Extensively drug-resistant Pseudomonas aeruginosa (XDR-PA) is a life-threatening pathogen that causes serious global problems. Here, we investigated two novel P. aeruginosa bacteriophages (phages), Bϕ-R656 and Bϕ-R1836, in vitro, in silico, and in vivo to evaluate the potential of phage therapy to control XDR-PA clinical strains. Bϕ-R656 and Bϕ-R1836 belong to the Siphoviridae family and exhibited broad host ranges which could lyse 18 (64%) and 14 (50%) of the 28 XDR-PA strains. In addition, the two phages showed strong bacteriolytic activity against XDR-PA host strains from pneumonia patients. The whole genomes of Bϕ-R656 and Bϕ-R1836 have linear double-stranded DNA of 60,919 and 37,714 bp, respectively. The complete sequence of Bϕ-R656 had very low similarity to the previously discovered P. aeruginosa phages in GenBank, but phage Bϕ-R1836 exhibited 98% and 91% nucleotide similarity to Pseudomonas phages YMC12/01/R24 and PA1/KOR/2010, respectively. In the two in vivo infection models, treatment with Bϕ-R656 and Bϕ-R1836 enhanced the survival of Galleria mellonella larvae (50% and 60%, respectively) at 72 h postinfection and pneumonia-model mice (66% and 83%, respectively) at 12 days postinfection compared with untreated controls. Treatment with Bϕ-R656 or Bϕ-R1836 also significantly decreased the bacterial load in the lungs of the mouse pneumonia model (>6 log10 CFU and >4 log10 CFU, respectively) on day 5.IMPORTANCE In this study, two novel P. aeruginosa phages, Bϕ-R656 and Bϕ-R1836, were evaluated in vitro, in silico, and in vivo for therapeutic efficacy and safety as an alternative antibacterial agent to control XDR-PA strains collected from pneumonia patients. Both phages exhibited potent bacteriolytic activity and greatly improved survival in G. mellonella larva infection and a mouse acute pneumonia model. Based on these results, we strongly predict that these two new phages could be used as fast-acting and safe alternative biological weapons against XDR-PA infections.ope

In Vivo Application of Bacteriophage as a Potential Therapeutic Agent To Control OXA-66-Like Carbapenemase-Producing Acinetobacter baumannii Strains Belonging to Sequence Type 357

The increasing prevalence of carbapenem-resistant Acinetobacter baumannii (CRAB) strains in intensive care units has caused major problems in public health worldwide. Our aim was to determine whether this phage could be used as an alternative therapeutic agent against multidrug-resistant bacterial strains, specifically CRAB clinical isolates, using a mouse model. Ten bacteriophages that caused lysis in CRAB strains, including blaOXA-66-like genes, were isolated. YMC13/01/C62 ABA BP (phage B?-C62), which showed the strongest lysis activity, was chosen for further study by transmission electron microscopy (TEM), host range test, one-step growth and phage adsorption rate, thermal and pH stability, bacteriolytic activity test, genome sequencing and bioinformatics analysis, and therapeutic effect of phage using a mouse intranasal infection model. The phage B?-C62 displayed high stability at various temperatures and pH values and strong cell lysis activity in vitro The phage B?-C62 genome has a double-stranded linear DNA with a length of 44,844 bp, and known virulence genes were not identified in silico. In vivo study showed that all mice treated with phage B?-C62 survived after intranasal bacterial challenge. Bacterial clearance in the lung was observed within 3 days after bacterial challenge, and histologic damage also improved significantly; moreover, no side effects were observed. IMPORTANCE: In our study, the novel A. baumannii phage B?-C62 was characterized and evaluated in vitro, in silico, and in vivo These results, including strong lytic activities and the improvement of survival rates, showed the therapeutic potential of the phage B?-C62 as an antimicrobial agent. This study reports the potential of a novel phage as a therapeutic candidate or nontoxic disinfectant against CRAB clinical isolates in vitro and in vivo.ope

Complete genome sequence of the podoviral bacteriophage YMC/09/02/B1251 ABA BP, which causes the lysis of an OXA-23-producing carbapenem-resistant Acinetobacter baumannii isolate from a septic patient

The emergence of carbapenem-resistant Acinetobacter baumannii, responsible for causing nosocomial infections, has been becoming a significant global health issue. In this article, we report the complete genome sequence of bacteriophage B-B1251 (YMC/09/02/B1251 ABA BP), which causes lysis of a carbapenem-resistant A. baumannii strain. The bacteriophage belongs to the family Podoviridae and has a double-stranded circular DNA genome with a length of 45,364 bp and a 39.05% G+C content. Genome analysis showed that it had no similarity to other previously reported bacteriophages capable of infecting A. baumannii.ope

Characterization and complete genome sequence analysis of two Myoviral bacteriophages infecting clinical carbapenem-resistant Acinetobacter baumannii isolates

AIMS: The aim of this study was to characterize phenotypical properties, to analyse whole genomes of novel Acinetobacter baumannii phages infecting carbapenem-resistant Ac. baumannii (CRAB) and to evaluate their potential as antimicrobial alternatives to control Ac. baumannii in clinical settings. METHODS AND RESULTS: The Ac. baumannii phages, Βϕ-R1215 and Βϕ-R2315, were isolated from sewage samples. These phages were characterized by transmission electron microscopy, host spectrum, the thermal/pH stability test, the bacterial lysis assay and the whole genome analysis. Both phages lysed 21 of 45 CRAB hosts, and showed high stability at various pH (pH 4-10) and temperature (25-60°C), and were strongly active against host bacteria in vitro. The genomes of Βϕ-R1215 and Βϕ-R2315 are linear double-strands of DNA with 44·866 and 44·846 bp respectively. These two genomes revealed high similarity at the DNA level, but the organization and direction of open reading frames were different. CONCLUSIONS: The Ac. baumannii phages, Βϕ-R1215 and Βϕ-R2315, are novel lytic phages lysing CRAB strains which were isolated from respiratory samples of patients. SIGNIFICANCE AND IMPACT OF THE STUDY: In vitro and in silico data showed that these novel Ac. baumannii phages, Βϕ-R1215 and Βϕ-R2315, have potential as antimicrobial alternatives to control CRAB in healthcare settings.ope

공공정보 민간 개방 및 활용 지원정책

행사명 : 한국지역정보화학회, 한국정보과학회, 국가DB포럼 공동 워크

Lytic phage of carbapenem-resistant Acinetobacter spp. as an alternative therapeutic agent : purification, whole genome analysis, safety and efficacy testing

의과대학/박사In recent years, antimicrobial resistance has become a major medical threat worldwide. Among them the rapid increase in carbapenem-resistant Acinetobacter baumannii (CRAB) is one of the particular global issues challenging the health care setting. In order to overcome the problems, bacteriophages are newly reviewed to be an alternative strategy for the control of these pathogens. In this study, we isolated and purified 15 A. baumannii phages, which cause lysis of CRAB strains, from sewage water at a hospital in South Korea, and six of the A. baumannii phages, phage YMC13/01/C62 ABA BP (phage Bphi-C62), YMC 13/01/R2096 ABA BP (phage Bphi-R2096), YMC11/12/R1215 ABA BP (phage Bphi-R1215), YMC11/12/R2315 ABA BP (phage Bphi-R2315), YMC/09/02/B1251 ABA BP (phage Bphi-B1251) and YMC11/11/R3177 ABA BP (phage Bphi-R3177), were studied. Characterization of the all phages was conducted by transmission electron microscopy (TEM), host range, adsorption rate, one-step growth curve, temperature/pH stability test and host cell lytic activity test against each host bacteria. Morphological properties of phages by TEM revealed that the A. baumannii phage Bphi-C62, Bphi-R2096, Bphi-R1215, Bphi-R2315 and Bphi-R3177 belong to the family Myoviridae and only phage Bphi-B1251 belongs to the family Podoviridae. In the characterization test of the phages, almost all the A. baumannii phages exhibited a broad spectrum against clinical CRAB isolates used in this study, but they did not lyse other bacterial strains including Gram-positive strains. These phages showed high absorption rate (> 95% within 5 min) and bust size (42-286 PFU/cell), and exhibited broad stability at various temperatures (25-70°C) and pH (pH 4-10). All phages showed strong host cell lytic activities at high-dose (MOI = 10). Complete genomes of all phages were sequenced using the 454-Junior Genome Analyzer. The phages have a double-stranded DNA with a length of approximately 44-47 kbp and 37-39% G+C content, however, genome of phage Bphi-R2096 had 98,170 bp, and lysogenic- or toxin-related genes were not identified in these phage genomes. Bioinformatics analysis showed that the whole genome sequence of phage Bphi-R2096 and Bphi-B1252 had no similarity to other previously reported bacteriophages capable of infecting A. baumannii. To investigate antibacterial activities of isolated phages against CRAB infection in vivo, we selected two A. baumannii phages, phage Bphi-C62 and Bphi-R2096, which exhibited strong host cell lytic activity in vitro. We evaluated their survival rate, histologic features, bacterial clearance and phage titers, and proinflammatory cytokines in the mouse lung infection model. The phage Bphi-62and Bphi-R2096 improved mouse survival rate and histologic damages of lung, and showed the bacterial clearance in the lung within 3 days after intranasal bacterial challenge. Also, no mortality or serious side effects, such as abnormal physical conditions, signs of toxicity, were observed in the phage-administrated mouse group or high-dose group (1011 PFU/ml) of phage. In summary, 6 novel A. baumannii phages -phage Bphi-C62, Bphi-R2096, Bphi-R1215, Bphi-R2315, Bphi-B1251 and Bphi-R3177- infecting clinical carbapenem-resistant A. baumannii strains were isolated and studied in detail on physiological characterizations and whole genome sequence analysis. Overall, the results revealed that these phages have a potential as an antimicrobial agent to control the spread of carbapenem-resistant A. baumannii strains in clinical settings. Especially, the in vivo results suggest that the novel A. baumannii phage Bphi-62and phageBphi-R2096 can be considered as a therapeutic agent for infectious disease caused by CRAB strains. To the best our knowledge, this is the first report of the characterization of A. baumannii phages in determining its potential as an alternative therapeutic agent using mouse model of pulmonary infection with CRAB clinical isolates.prohibitio

Complete genome sequence of the siphoviral bacteriophage B/-R3177, which lyses an OXA-66-producing carbapenem-resistant Acinetobacter baumannii isolate

In recent years, antimicrobial resistance has become a major medical threat worldwide. Among these threats, the rapid increase in carbapenem-resistant Acinetobacter baumannii (CRAB) is a particularly challenging global issue in the health care setting. In this study, a novel lytic A. baumannii phage, B/-R3177, infecting carbapenem-resistant A. baumannii strains was isolated from sewage samples at a hospital. The morphology of the phage as assessed by transmission electron microscopy (TEM) indicated that it belongs to the family Siphoviridae within the order Caudovirales. It has a linear double-stranded DNA genome of 47,575 bp with a G?C content of 39.83 %. Eighty open reading frames (ORFs) were predicted; however, only 14 ORFs were annotated as encoding functional proteins, while most of the ORFs encoded hypothetical proteins. Among the total ORFs of the phage genome, no toxin-related genes were detected. A bioinformatics analysis showed that the whole genome sequence of phage B/-R3177 exhibited 62 % sequence similarity to that of Acinetobacter phage B/-B1252, but there was no homology seen with other phages. Physiological characteristics, such as one-step growth properties, pH and temperature stability, and host cell lysis activity showed this phage has high stability and lytic activity against host bacteria and therefore has potential applicability as an antibacterial agent to control pathogens in the hospital environment.restrictio

Complete genome sequence of the bacteriophage YMC01/01/P52 PAE BP, which causes lysis of verona integron-encoded metallo-β-lactamase-producing, carbapenem-resistant Pseudomonas aeruginosa which causes lysis of verona integron-encoded metallo-beta-lactamase-producing, carbapenem-resistant pseudomonas aeruginosa

Multidrug-resistant Pseudomonas aeruginosa commonly causes serious nosocomial infections. In this study, a novel lytic bacteriophage belonging to a member of the family Podoviridae, YMC01/01/P52 PAE BP, which infects carbapenem-resistant Pseudomonas aeruginosa, was isolated and characterized. YMC01/01/P52 PAE BP genome was analyzed by whole-genome sequencing and putative function identification. The bacteriophage genome consists of a double-stranded linear DNA genome of 49,381 bp with a GC content of 62.16%.ope

Complete Genome Sequence of the Siphoviral Bacteriophage YMC/09/04/R1988 MRSA BP: A lytic phage from a methicillin-resistant Staphylococcus aureus isolate.

Methicillin-resistant Staphylococcus aureus (MRSA) is an increasing cause of serious infection, both in the community and hospital settings. Despite sophisticated strategies and efforts, the antibiotic options for treating MRSA infection have been narrowed due to the limited number of newly developed antimicrobials. Herein, we analyze the completely sequenced genome of a novel virulent phage YMC/09/04/R1988 MRSA BP as a potential alternative anti-MRSA agent, which lysed clinical isolates from a patient admitted to the hospital due to hip disarticulation. The phage contains a linear double-stranded DNA genome of 44,459 bp in length, with 33.37% GC content, 62 predicted open reading frames (ORFs), and annotated functions of only 23 ORFs that are associated with structural assembly, host lysis, DNA replication, and modification. It showed a broad host range (17 of 30 strains) against MRSA strains in clinical isolates.ope