The Computational Diet: A Review of Computational Methods Across Diet, Microbiome, and Health.

Food and human health are inextricably linked. As such, revolutionary impacts on health have been derived from advances in the production and distribution of food relating to food safety and fortification with micronutrients. During the past two decades, it has become apparent that the human microbiome has the potential to modulate health, including in ways that may be related to diet and the composition of specific foods. Despite the excitement and potential surrounding this area, the complexity of the gut microbiome, the chemical composition of food, and their interplay in situ remains a daunting task to fully understand. However, recent advances in high-throughput sequencing, metabolomics profiling, compositional analysis of food, and the emergence of electronic health records provide new sources of data that can contribute to addressing this challenge. Computational science will play an essential role in this effort as it will provide the foundation to integrate these data layers and derive insights capable of revealing and understanding the complex interactions between diet, gut microbiome, and health. Here, we review the current knowledge on diet-health-gut microbiota, relevant data sources, bioinformatics tools, machine learning capabilities, as well as the intellectual property and legislative regulatory landscape. We provide guidance on employing machine learning and data analytics, identify gaps in current methods, and describe new scenarios to be unlocked in the next few years in the context of current knowledge

Evolution of Animal Microbial Communities in Response to Environmental Stress

info:eu-repo/semantics/publishedVersio

METAGENOMIC INSIGHTS INTO MICROBIAL COMMUNITY RESPONSES TO LONG-TERM ELEVATED CO2

Understanding how belowground microbial communities respond to increasing atmospheric CO2 is of crucial importance for global change biology, microbial ecology and predictive biology. However, our understanding of CO2 effects on microbial communities is still limited, especially due to the immense diversity and as yet-uncultivable nature of most soil microorganisms. By implementing next generation sequencing (NGS) technologies, we comprehensively surveyed the responses of microbial communities to elevated CO2 (eCO2) in the BioCON experimental site, a grassland ecosystem, which had been exposed to eCO2 for 12 years. In the beginning of this study, it was noticed that computational approaches to identify microbial strains/species from shotgum metagenomes are very limited, thus we have developed a computational algorithm, termed GSMer that identifies genome-specific markers (GSMs) from currently sequenced microbial genomes for strain/species identification in metagenomes. Although GSMer was not very successfully applied in our soil metagenomes due to the extremely low coverage and high diversity of soil microbial communities as well as short sequencing reads from early Illumina GAII performs, it was successfully used to analyze microbial communities with a good coverage of reference genomes, such as human microbiomes. Sensitivity evaluation against synthetic metagenomes with different levels of coverage suggested that 50 GSMs per strain were sufficient to identify most microbial strains with ≥ 0.25x coverage, and 10% of selected GSMs in a database should be detected for confident positive callings. Application of GSMs respectively identified 45 and 74 microbial strains/species significantly associated with type-2-diabetes (T2D) patients and obese/lean individuals from corresponding gastrointestinal tract metagenomes. Our results agreed well with previous studies, but provided strain-level information. In the following, we analyzed the biodiversity, composition, structure and functional potential of soil microbial communities in response to eCO2 at multiple (e.g., phylogenetic, taxonomic, genetic, functional) dimensions using next generation sequencing approaches. For each dimension of microbial biodiversity, all components of diversity, including alpha-, beta- and gamma-diversity were analyzed. Our results suggested that long-term eCO2 decreased the overall microbial biodiversity. Beta-diversity analysis suggested eCO2 decreased functional beta-diversity, but increased taxonomic and phylogenetic diversity, suggesting long-term eCO2 selected for microbial function rather than taxonomy. Further meta-analysis suggested that such decreased biodiversity was significantly negatively correlated with increased soil ammonification rate. Moreover, the abundance of gene families involved in ammonium producing pathways increased significantly as well, indicating a functional convergence process as a result of higher demand for biologically available nitrogen (N) by stimulated plant growth as a result of eCO2. Our findings present evidence that plant-microbe interactions for NH4+ as result of progressive nitrogen limitation were an important driving factor, responsible for decreased microbial biodiversity under eCO2. We also analyzed the response of fungal communities to long-term eCO2 by sequencing of 28S rRNA gene amplicons. Long-term eCO2 did not significantly alter the overall fungal community structure and species richness, but significantly increased community evenness and diversity. Relative abundances of 119 OTUs (~ 27% of the total captured sequences) were changed significantly. More interestingly, significantly changed OTUs under eCO2 were associated with increased overall relative abundance of Ascomycota, but decreased relative abundance of Basidiomycota. Co-occurrence ecological network analysis indicated that eCO2 increased fungal community interactions, as evidenced by higher intermodular and intramodular connectivity and shorter geodesic distance. In contrast, decreased connections for dominant fungal species were observed in the eCO2 network. Community reassembly of unrelated fungal species into highly connected dense modules was observed. Such changes in the network structure were significantly associated with altered soil and plant properties under eCO2, especially with increased plant biomass and NH4+ availability. This study provides novel insights into our understanding of how eCO2 shapes soil fungal communities in grassland ecosystems. Since it was noticed that changes of both belowground microbial biodiversity and fungal communities were significantly correlated with soil ammonification rate, and our previous studies showed that the abundance of nifH significantly increased at eCO2, we hypothesized that N2-fixing microorganisms would play important roles in response to eCO2. Therefore, we analyzed N2-fixing communities by sequencing of nifH gene amplicons as well as extraction of nifH fragments from shotgun metagenomes. Surprisingly, long-term eCO2 significantly increased the abundance of nifH genes, but did not change the overall nifH diversity and diazotrophic community structure. Taxonomic and phylogenetic analysis of amplified nifH sequences suggested a high diversity of nifH genes in the soil ecosystem, with the majority belonging to cluster I and II nifH genes. We then constructed a microbial ecological network using 16S rRNA gene and nifH gene profiles. Co-occurrence ecological network analysis suggested a clear preference of co-occurrence patterns between diazotrophs and other microbial species with different patterns observed for different subgroups of diazotrophs, such as Azospirillum/Actinobacteria, Mesorhizobium/Conexibacter, and Bradyrhizobium/Acidobacteria. This indicated a potential attraction of these non-N2-fixers by diazotrophs in the soil ecosystem. Interestingly, more complex co-occurrence patterns were found for free-living diazotrophs than commonly known symbiotic diazotrophs, consistent with the physical isolation nature of symbiotic diazotrophs from the environment by root nodules. The study provides novel insights of our understanding microbial ecology of soil diazotrophs in natural ecosystems. All studies included in this work provided novel insights into the long-term eCO2 effects on belowground microbial communities, which are of merit for next generation sequencing analysis for microbial ecologists, global change biologists and bioinformaticians

A natural mutation in Pisum sativum L. (pea) alters starch assembly and improves glucose homeostasis in humans

Elevated postprandial glucose (PPG) is a significant risk factor for non-communicable diseases globally. Currently, there is a limited understanding of how starch structures within a carbohydrate-rich food matrix interact with the gut luminal environment to control PPG. Here, we use pea seeds (Pisum sativum) and pea flour, derived from two near-identical pea genotypes (BC1/19RR and BC1/19rr) differing primarily in the type of starch accumulated, to explore the contribution of starch structure, food matrix and intestinal environment to PPG. Using stable isotope 13C-labelled pea seeds, coupled with synchronous gastric, duodenal and plasma sampling in vivo, we demonstrate that maintenance of cell structure and changes in starch morphology are closely related to lower glucose availability in the small intestine, resulting in acutely lower PPG and promotion of changes in the gut bacterial composition associated with long-term metabolic health improvements

Betydning av kostholdsfaktorer og reaktive oksygenforbindelser (ROS) for tarmen og dens mikrobiota i en musemodell med lavgrads inflammasjon

Background: Various factors, including dietary components, the gut microbiota and host responses, are imperative for gut health. Optimal interactions between diet, gut microbiota and host are suggested to be important to avoid imbalanced gut microbial ecosystems and low-grade inflammation. Dietary fiber components, in particular, have been highlighted for their positive effects on the microbiota and gut inflammation status. Furthermore, although high-fat diets, mainly based on animal fat, have been associated with gut inflammation it is not clear to what extend different fat sources (i.e., animal type), and modes of fat intake affect the gut inflammatory process. Objectives: The overall aim of the current thesis was to investigate the differential impact of fiber and protein fractions from faba beans and different fat types on the microbiota composition and gut health in mice with and without low-grade inflammation in the gut. Another aim was to assess the role of reactive oxygen species (ROS) induced by NADPH oxidase 1 (NOX1) for low-grade gut inflammation and microbial community in the colon. Methods: After the establishment of a low-grade inflammation model with the chemical dextran sodium sulfate (DSS) feeding trials with C57BL/6J mice were performed. Besides the wild type (WT), C57BL/6J, mice deficient in the NOX1 gene were used (NOX1-KO mice). The diets offered during the mouse experiments varied in macronutrient composition and energy intake. Briefly these diets were: a standard mouse diet (chow diet), a synthetic low-fat diet (LFD), a purified western diet (WD) and modified western diets (WDs), in which defined fractions from faba beans were incorporated. These fractions were protein fraction (PF) and fiber fraction (FF) that replaced partially casein and cellulose substances in WDs, respectively. Body weight, colon length, gene expression of inflammatory and reactive oxygen species related genes, biomarkers of inflammation in blood and fecal sample were parameters to be examined. Shifts of the colonic microbiota populations in phylum and genus level upon the termination of experiments were additionally evaluated using LEfSe (Linear discriminant analysis Effect Size). Results: In Paper I, we established the low-grade inflammation mouse model and showed that NOX1 plays a significant role in ROS formation in the colon during pathogenesis towards the status of low-grade inflammation. We demonstrated that NOX1 modulates the colonic microbiota both in a steady-state (healthy) and during low grade inflammation. Also, while absence of NOX-1 did not influence the pathology scores, the inflammation-related genes and lipocalin 2 (LCN-2) expression tended to be higher in NOX1 deficient mice than in WT mice. From these results we conclude that NOX1-dependent ROS production is important for shaping microbiota composition and for protecting against insults to the colon. In Paper II, we compared the impact of a low-fat diet (LFD) and different western diets (WD) with fat from various sources on colon health in low-grade inflammation. We falsified the hypothesis that WD fed mice would manifest more severe symptoms compared to LFD fed mice as we observed that LFD mice were more susceptible to DSS-induced inflammation and revealed a less diverse microbiota with increased relative abundance of the Proteobacteria phylum compared to WD fed mice. Both fat sources (milk and lard) tested as part of the WD induced similar immediate ‘protective’ effect as compared to the LFD. Because these results contradict many other studies, we suspect that the higher fat content protects against the establishment of the DSS-induced low-grade inflammation. Therefore, we raise the concern that the DSS model of mouse inflammation might be unfit for the purpose of studying the differential impact of dietary fats. In Paper III, we investigated possible metabolic and colonic beneficial effects of faba bean protein and fiber fractions when ingested as part of a WD. Although the diets were isocaloric the mice that ingested diets with added faba bean protein content (WD+PF and WD+BF) had a higher increase in body weight compared to the pure WD or the WD+FF. However, no differences were revealed in glucose and insulin tolerance test between dietary groups. Also, neither the protein nor the fiber fraction protected against DSS induced low-grade gut inflammation as compared with mice fed a pure WD. Furthermore, marginal changes were found in the microbiota at the genus level. Conclusions: Collectively, our results demonstrate a role for ROS in the healthy gut and in response to DSS induced inflammation which may be related to controlling and shaping the microbiota. Furthermore, unexpectedly, we found that a high fat content in the diet protected against DSS induced inflammation as compared with the LFD group and speculate that the high fat content confounds the establishment of the DSS induced low-grade inflammation model. Also, a WD with a higher protein fraction from faba bean resulted in even higher gain of weight compared to standard WD. Only marginal changes were found for the microbiota composition when increasing the fiber fraction of the WD and it did not protect against weight gain.Bakgrunn: Ulike faktorer, slik som næringsstoffer, tarmmikrobiota og biologiske vertsresponser, er avgjørende for tarmhelsen. Optimal interaksjon mellom disse er viktig for å unngå ubalanserte mikrobielle tarmøkosystemer og lavgradig betennelse. Særlig har kostfiber blitt fremhevet som viktig og positiv for både mikrobiota og tarmbetennelsesstatus. I tillegg, selv om kosthold med høyt fettinnhold fra animalske kilder har vært assosiert med tarmbetennelse, er det ikke entydig klart i hvilken grad ulike fettkilder (dvs. dyretype), og inntak av fett som sådan påvirker betennelse i tarm. Mål: Det overordnede målet med denne oppgaven var å undersøke i hvilken grad fiber- og proteinfraksjoner fra fababønner og ulike fetttyper påvirket mikrobiotasammensetningen og tarmhelsen hos mus med og uten lavgradig betennelse i tarmen. Et annet mål var å evaluere rollen til reaktive oksygenforbindelser (ROS) indusert av NADPH-oksidase 1 (NOX1) for lavgradig tarmbetennelse og mikrobielle samfunn i tykktarm. Metoder: Etter etablering av en lavgradig inflammasjonsmodell med det kjemiske stoffet dekstran-natriumsulfat/dextran sodium sulfate (DSS) ble det utført fôringsforsøk med C57BL/6J-mus. I tillegg til å bruke villtypemus (WT), ble C57BL/6J-mus uten uttrykk av NOX1-genet benyttet (NOX1-KO-mus). De ulike fôrtypene som ble brukt under museforsøkene varierte i sammensetning av makronæringsstoffer og energiinnhold. Fôrtypene bestod (i korthet) av: standard musefôr også kalt chow, lav-fettdiett (LFD), en vestlig høy-fettdiett (WD) og modifiserte vestlige dietter, der fraksjoner fra faba bønner ble inkorporert. Disse fraksjonene var proteinfraksjon (PF) og fiberfraksjon (FF) som delvis erstattet henholdsvis kasein- og cellulose i WD-fôrtypene. Alle fôrtypene med unntak av chow var kontrollerte (kjent innhold av alle næringsstoffene og ingrediensene). Chow-fôret er mer udefinert fordi det er satt sammen av hele råvarer. Kroppsvekt, tykktarmslengde, genuttrykk av inflammatoriske og ROS-relaterte gener, biomarkører for betennelse i blod og avføringsprøver var parametere som skulle undersøkes. Endringer av tykktarmens mikroflora ved avslutning av eksperimenter ble i tillegg evaluert ved bruk av såkalt LefSe (Linear discriminant analysis Effect size). Resultater: I Artikkel I (Paper I) etablerte vi musemodell for lavgradig inflammasjon og viste at NOX1 spiller en betydelig rolle i ROS-produksjon i tykktarmen under lavgradig betennelse indusert med DSS. Vi viste også at NOX1 modulerer tykktarmsmikrobiotaen både i en ‘steady-state’ (sunn) og under lavgradig betennelse. Videre så vi at, mens fravær av NOX-1 ikke påvirket patologi, hadde de betennelsesrelaterte genene og uttrykk av lipocalin 2 (LCN-2) en tendens til å være høyere i NOX1-knockout mus enn i WT-mus. Fra disse resultatene konkluderer vi med at NOX1-avhengig ROS-produksjon er viktig for å forme mikrobiotasammensetning og for å beskytte mot skader i tykktarmen. I Artikkel II (Paper II) sammenlignet vi effekten av en lav-fettdiett (LFD) og ulike vestlige dietter (WD) med fett fra ulike kilder på tykktarmshelsen ved lavgradig betennelse. Vi forkastet derfor hypotesen om at WD-fôrede mus ville manifestere mer alvorlige symptomer sammenlignet med LFD-fôrede mus da vi observerte at LFD-mus var mer mottakelige for DSS-indusert betennelse og avslørte en mindre mangfoldig mikrobiota med økt relativ tilstedeværelse av Proteobakterier (fylum) sammenlignet med WD-fôrede mus. Begge fettkilder (melk og svinefett) testet som en del av WD førte i begge tilfeller til umiddelbar "beskyttende" effekt sammenlignet med LFD. Fordi disse resultatene motsier mange andre studier, mistenker vi at det høyere fettinnholdet beskytter mot etableringen av DSS-indusert lavgradig betennelse. Derfor reiser vi bekymringen for at DSS-modellen for musebetennelse kan være uegnet for det formål å studere den differensielle påvirkningen av fettrikt fôr med den hensikt å studere fysiologiske effekter av høyfett-dietter. I Artikkel II (Paper III) undersøkte vi potensielt metabolske effekter av proteiner og fiber (begge var fraksjoner) isolert fra fababønner som ble blandet inn WD-fôret. Selv om diettene var isokaloriske, hadde musene som inntok fôret beriket med proteiner fra fababønner (WD+PF og WD+BF) fant vi at disse musene hadde en høyere økning i kroppsvekt sammenlignet med ren WD eller WD+FF (fiberfraksjon). Imidlertid ble det ikke avdekket forskjeller i glukose- og insulinregulering mellom de ulike diettgruppene. Heller ikke protein- eller fiberfraksjonen fra fababønner beskyttet mot DSS induserte lavgradig tarmbetennelse sammenlignet med mus som ble fôret med en ren WD. Videre ble det funnet marginale endringer i mikrobiotaen på slektsnivå. Konklusjoner: Samlet viser resultatene våre en rolle for ROS i den sunne tarmen og som respons på DSS-indusert betennelse som kan være relatert til å kontrollere og forme mikrobiotaen. Videre, og noe uventet, fant vi at et høyt fettinnhold i kostholdet (WD) beskyttet mot DSS-indusert betennelse sammenlignet med LFD-gruppen og spekulerer i at det høye fettinnholdet påvirker etableringen av den DSS-induserte lavgradige betennelsesmodellen, muligens direkte. Dessuten resulterte en WD med en høyere proteinfraksjon fra fababønne i enda høyere vektøkning sammenlignet med standard WD. Bare marginale endringer ble funnet for mikrobiotasammensetningen ved tilsetning av fiberfraksjonen til WD, og den beskyttet ikke mot vektøkning

16S rRNA 메타 유전체 연관성 분석 통계 방법론의 개발과 적용

학위논문(박사) -- 서울대학교대학원 : 보건대학원 보건학과(보건학전공), 2021.8. 원성호.연구배경: 시퀀싱 기술의 발달과 시퀀싱 비용 감소는 미생물 군집에 대한 대규모 분석을 가능하게 하였고 메타 유전체학이 탄생하였으며 이 분야가 광범위하게 발전하였다. 구성 편향과 제로 팽창 문제는 메타 게놈 데이터의 연관 분석을 위한 통계적 방법도 수행하기 어렵게 만든다. 또한 이러한 문제는 반복 측정 내에서 복잡한 상관 관계를 고려해야하는 종단 분석의 모델링을 더 어렵게 만든다. 이러한 희박함과 다양한 데이터베이스 및 클러스터링 방법 선택은 미생물 군유 전체 데이터 세트의 이질성을 유도한다. 연구목적: 이 연구의 목적은 (1) 다양한 클러스터링 방법과 데이터베이스를 기반으로 결과를 비교할 수있는 구성 편향, 제로 인플레이션, 패키지 구현 등 문제를 수정하는 통계적 방법을 개발하는 것이다. (2) 구성 편향, 제로 인플레이션, 종단 데이터 세트 반복 측정 간의 상관 관계 등 문제를 수정하는 통계적 방법 개발, (3) 제 2형 당뇨병 위험 지표에 영향을 줄 수 있는 미생물을 식별하고 다중 오믹스 자료를 활용한 종단 연관분석을 통하여 이를 설명하는 생물학적 배경을 발견한다. 연구방법: 미생물 군유 전체 데이터의 특성을 수정하고 구성 편향 및 제로 팽창 문제를 수정하기 위해 풍부도를 정규화하고 트리 참조 트리 구조와 결합합니다. 전처리 절차와 다른 데이터베이스와의 결과 비교 및 클러스터링 방법을 포함하는 패키지가 개발되어 이질성 문제를 처리 할 수 있습니다. 반복 측정 값 간의 상관 관계는 각각 로버 스트 점수와 Wald 통계를 사용하여 일반화 된 추정 방정식을 반영한다. 제 2 형 당뇨병 위험 지표는 일반화 된 추정 방정식이있는 모델이며 생물학적 메커니즘은 추정 된 기능 게놈 및 SNP를 통해 탐색되었다. 목표 미생물과 제 2 형 당뇨병 위험 사이의 인과 관계를 조사하기 위해 Mendelian 무작위 분석도 수행되었다. 연구 결과 및 결론: 계통 발생 트리 기반 미생물 군집 연합 테스트 (TMAT)는 미생물 풍부도를 표준화하고 계통 발생 트리 구조와 결합하였다. 계통 발생 수를 기반으로 한 시퀀싱 판독의 통합은 제로 인플레이션을 줄이고 두 미생물 풍부 사이의 비율을 취하면 구성 편향을 수정하였다. 다양한 데이터베이스와 클러스터링 방법을 기반으로 한 파이프 라인 구축 미생물 수표를 포함하는 패키지 인 포괄적 인 미생물 군유 전체 연관 분석 (AMAA)과 메타 게놈 전체 연관 분석 방법을 개발하였으며 이를 통해 다양한 데이터베이스 또는 클러스터링 방식을 기반으로 한 통합 전처리 및 결과 비교를 통해 다양한 미생물 군유 전체 연관성 분석 방법을 편리하게 사용할 수 있을 것이다. TMAT의 확장 버전 mTMAT는 강력한 분산 추정기를 사용하며 반복 측정 된 샘플에 적용 할 수 있다. mTMAT의 통계적 파워는 명목 유형 1 오류를 보존하는 대부분의 시나리오에서 다른 방법보다 우수하였다. 우리는 Lachnospiraceae 계통의 GU174097이 제 2 형 당뇨병 위험 지표와 상관 관계가 있음을 발견하였다. 또한 이 속은 단쇄 지방산 (SCFA)과 관련된 경로와 관련이있을 수 있음이 밝혀져 있으며 MR 분석 및 생물학적 배경 조사는 이 속이 당뇨의 위험을 증가시킬 수 있다는 가능성을 시사한다.Background: Increased availability of affordable sequencing technology and advances in throughput technology have led to the birth and widespread development of a new scientific discipline, metagenomics that includes large-scale analysis of microbial communities. However, analysis with metagenomics data suffers from compositional bias and zero-inflated problems, and the statistical methods available for association analysis with 16S rRNA data is very limited, especially for the repeatedly observed 16S rRNA data. Therefore investigation on the statistical method and software development is necessary. Objective: The main goal is (1) to develop new methods with cross-sectional and repeatedly observed 16S rRNA data that correct for the problems including compositional bias, zero-inflation and package implementation that can unify the preprocessing procedures; (2) to identify microorganisms which can be affect type-2 diabetes (T2D)-related traits with repeatedly observed 16S rRNA data. Methods: To consider the characteristics of microbiome data and correct compositional bias and zero-inflated problem, the phylogenetic tree based method, TMAT, and its extension to the repeatedly observed 16S rRNA measurement, mTMAT, were developed. I also implemented a new package that can generate both statistics, and conduct OTU clustering with different databases. This package also allows the comparison of different statistics. Furthermore, association analysis of microorganisms with T2D were conducted by using repeatedly measured EV in urine samples. EV-derived metagenomic (N = 393), clinical (N = 5032), and metabolite (N = 574) data were observed for a prospective and longitudinal Korean community-based cohort (KARE) three times and genetic data was available. They were analyzed with generalized linear mixed model to identify microbes associated with T2D and their interaction with metabolites. Results and Conclusions: The proposed phylogenetic tree-based microbiome association test (TMAT) normalized microbial abundances and pooled abundances based on the phylogenetic tree structure was utilized for association analysis. Results from simulation studies showed that TMAT correctly controls type-1 error rates, and statistically more powerful. Second, I also implemented all-inclusive microbiome association analysis (AMAA) package. AMAA package provides the analysis result of various methods including TMAT under a unified preprocessing and allows comparison of the results based on different databases or clustering methods. Third, mTMAT which is the extended version of TMAT for repeatedly measured 16S rRNA data was developed. It uses generalized estimating equations with robust variance estimator and can be applied to repeated measured samples. Statistical power of mTMAT was superior to existing methods in terms of controlling the type-1 error and minimizing the type-2 error, and it is robust against the compositional bias. Fourth, from the association analysis with repeatedly measured EV-based metagenome data, it was found that GU174097_g, an uncultured Lachnospiraceae, was associated with T2D (β = −189.13; p = 0.00006). These results indicates that GU174097_g may decrease the HbA1c level and the risk of T2D.Chapter 1. Introduction 8 1. Study Background 8 2. Literature Review 11 3. Purpose of Research 15 Chapter 2. Phylogenetic Tree-based Microbiome Association Test and Package Development for Microbiome Analysis 16 2.1 Introduction 16 2.2 Materials and Methods 19 2.3 Results 35 2.4 Discussion 55 Chapter 3. Longitudinal Microbiome Association Test based on Phylogenetic Tree 59 3.1 Introduction 59 3.2 Materials and Methods 62 3.3 Results 73 3.4 Discussion 110 Chapter 4. Longitudinal Measurement of Urine Microbiome Reveals the Role of uncultured Lachnospiraceae on Type-2 Diabetes Pathogenesis 112 4.1 Introduction 112 4.2 Materials and Methods 114 4.3 Results 126 4.4 Discussion 147 Chapter 5. Conclusions 150 References 152 Abstract in Korean 159박

Effects of sodium benzoate on the innate immune response to gram-negative bacteria and Toll-like receptor stimulation

Sodium benzoate (NaB) is a sodium salt that is widely used in the pharmaceutical, cosmetic and food industries. This widespread use results in almost everybody in the world being exposed daily to this compound. Currently very little is known about the effects of NaB can have on the immune response, even though it has been associated with the clinical course of chronic inflammatory diseases, such as orofacial granulomatosis and neurodegenerative disorders. Here, I show that THP-1 cells, a monocytic human cell line presents an altered immune response when exposed to NaB. Immunologically stimulated THP-1 cells in the presence of NaB secreted reduced levels of IL6 and IL1β and higher levels of TNF, while other cytokines such as IP10 and IL8 were unaffected. The inhibitory effect in IL6 and IL1β secretion was a consequence of a free radical scavenging characteristic of NaB, which neutralizes the reactive oxygen species (ROS) generated downstream of TLR activation. This resulted in the impairment of a secondary signalling event, which is required to fully activate the cells immune response. The use of microarray analysis in combination with q quantitative proteomic analysis revealed that NaB has a significant effect on the THP-1 cells beyond the alteration in cytokine secretion. NaB also interferes with cellular amino acid metabolism and has a major attenuating effect on the immune response. Taken together, these results suggest that NaB is not inert and has a major effect on a cells ability to mount an immune response. These findings could have major implications in how NaB is used in the future and in particular if it can be beneficial as a treatment for chronic inflammatory diseases, such as diabetes, atherosclerosis, rheumatoid arthritis, neurodegenerative disorders, and so on. On the other hand, by disturbing the inflammatory response, NaB could have a negative impact on other conditions such as orofacial granulomatosis. Further work will be needed to determine the role NaB plays in human inflammatory diseases

Saving Bones: a direct comparison of FTIR-ATR, whole bone percent nitrogen, and NIR

89th Annual Meeting of the American-Association-of-Physical-Anthropologists (AAPA), Los Angeles, CA, APR 15-18, 202