EBER2 RNA-induced transcriptome changes identify cellular processes likely targeted during Epstein Barr Virus infection

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Dietary soy and meat proteins induce distinct physiological and gene expression changes in rats

This study reports on a comprehensive comparison of the effects of soy and meat proteins given at the recommended level on physiological markers of metabolic syndrome and the hepatic transcriptome. Male rats were fed semi-synthetic diets for 1 wk that differed only regarding protein source, with casein serving as reference. Body weight gain and adipose tissue mass were significantly reduced by soy but not meat proteins. The insulin resistance index was improved by soy, and to a lesser extent by meat proteins. Liver triacylglycerol contents were reduced by both protein sources, which coincided with increased plasma triacylglycerol concentrations. Both soy and meat proteins changed plasma amino acid patterns. The expression of 1571 and 1369 genes were altered by soy and meat proteins respectively. Functional classification revealed that lipid, energy and amino acid metabolic pathways, as well as insulin signaling pathways were regulated differently by soy and meat proteins. Several transcriptional regulators, including NFE2L2, ATF4, Srebf1 and Rictor were identified as potential key upstream regulators. These results suggest that soy and meat proteins induce distinct physiological and gene expression responses in rats and provide novel evidence and suggestions for the health effects of different protein sources in human diets

Determining the impact of alternative splicing events on transcriptome dynamics

<p>Abstract</p> <p>Background</p> <p>The complete sequencing of the human genome and its subsequent analysis revealed a predominant role for alternative splicing in the generation of proteome diversity. Splice switching oligonucleotides (SSOs) are a powerful and specific tool to experimentally control alternative splicing of endogenous messenger RNAs in living cells. SSOs also have therapeutic potential to treat diseases that are caused by aberrant splicing. The assignment of biological roles to alternative splicing events of currently unknown function promises to provide a largely untapped source of potential new therapeutic targets. Here we have developed a protocol that combines high sensitivity microarrays with the transfection of SSOs to monitor global changes in gene expression downstream of alternate, endogenous splice events.</p> <p>Results</p> <p>When applied to a well-characterized splicing event in the Bcl-x gene, the application of high sensitivity microarrays revealed a link between the induction of the Bcl-xS isoform and the repression of genes involved in protein synthesis.</p> <p>Conclusion</p> <p>The strategy introduced herein provides a useful approach to define the biological impact of any given alternative splicing event on global gene expression patterns. Furthermore, our data provide the first link between Bcl-xS expression and the repression of ribosomal protein gene expression.</p

INTEGRATIVE ANALYSIS OF OMICS DATA IN ADULT GLIOMA AND OTHER TCGA CANCERS TO GUIDE PRECISION MEDICINE

Transcriptomic profiling and gene expression signatures have been widely applied as effective approaches for enhancing the molecular classification, diagnosis, prognosis or prediction of therapeutic response towards personalized therapy for cancer patients. Thanks to modern genome-wide profiling technology, scientists are able to build engines leveraging massive genomic variations and integrating with clinical data to identify “at risk” individuals for the sake of prevention, diagnosis and therapeutic interventions. In my graduate work for my Ph.D. thesis, I have investigated genomic sequencing data mining to comprehensively characterise molecular classifications and aberrant genomic events associated with clinical prognosis and treatment response, through applying high-dimensional omics genomic data to promote the understanding of gene signatures and somatic molecular alterations contributing to cancer progression and clinical outcomes. Following this motivation, my dissertation has been focused on the following three topics in translational genomics. 1) Characterization of transcriptomic plasticity and its association with the tumor microenvironment in glioblastoma (GBM). I have integrated transcriptomic, genomic, protein and clinical data to increase the accuracy of GBM classification, and identify the association between the GBM mesenchymal subtype and reduced tumorpurity, accompanied with increased presence of tumor-associated microglia. Then I have tackled the sole source of microglial as intrinsic tumor bulk but not their corresponding neurosphere cells through both transcriptional and protein level analysis using a panel of sphere-forming glioma cultures and their parent GBM samples.FurthermoreI have demonstrated my hypothesis through longitudinal analysis of paired primary and recurrent GBM samples that the phenotypic alterations of GBM subtypes are not due to intrinsic proneural-to-mesenchymal transition in tumor cells, rather it is intertwined with increased level of microglia upon disease recurrence. Collectively I have elucidated the critical role of tumor microenvironment (Microglia and macrophages from central nervous system) contributing to the intra-tumor heterogeneity and accurate classification of GBM patients based on transcriptomic profiling, which will not only significantly impact on clinical perspective but also pave the way for preclinical cancer research. 2) Identification of prognostic gene signatures that stratify adult diffuse glioma patientsharboring1p/19q co-deletions. I have compared multiple statistical methods and derived a gene signature significantly associated with survival by applying a machine learning algorithm. Then I have identified inflammatory response and acetylation activity that associated with malignant progression of 1p/19q co-deleted glioma. In addition, I showed this signature translates to other types of adult diffuse glioma, suggesting its universality in the pathobiology of other subset gliomas. My efforts on integrative data analysis of this highly curated data set usingoptimizedstatistical models will reflect the pending update to WHO classification system oftumorsin the central nervous system (CNS). 3) Comprehensive characterization of somatic fusion transcripts in Pan-Cancers. I have identified a panel of novel fusion transcripts across all of TCGA cancer types through transcriptomic profiling. Then I have predicted fusion proteins with kinase activity and hub function of pathway network based on the annotation of genetically mobile domains and functional domain architectures. I have evaluated a panel of in -frame gene fusions as potential driver mutations based on network fusion centrality hypothesis. I have also characterised the emerging complexity of genetic architecture in fusion transcripts through integrating genomic structure and somatic variants and delineating the distinct genomic patterns of fusion events across different cancer types. Overall my exploration of the pathogenetic impact and clinical relevance of candidate gene fusions have provided fundamental insights into the management of a subset of cancer patients by predicting the oncogenic signalling and specific drug targets encoded by these fusion genes. Taken together, the translational genomic research I have conducted during my Ph.D. study will shed new light on precision medicine and contribute to the cancer research community. The novel classification concept, gene signature and fusion transcripts I have identified will address several hotly debated issues in translational genomics, such as complex interactions between tumor bulks and their adjacent microenvironments, prognostic markers for clinical diagnostics and personalized therapy, distinct patterns of genomic structure alterations and oncogenic events in different cancer types, therefore facilitating our understanding of genomic alterations and moving us towards the development of precision medicine

대사 수술을 통한 포도당 항상성 호전의 기전

학위논문(박사)--서울대학교 대학원 :의과대학 의학과,2019. 8. 조영민.비만은 현대사회에서 가장 큰 의료 부담을 야기하는 질병의 하나로써 다양한 대사, 심혈관, 근골격 질환 그리고 종양과 연관되어 있다. 특히 제2형 당뇨병은 이러한 비만의 동반 질환 중에서 가장 대표적인 질병으로 환자들의 삶의 질 저하와 사망률의 증가로 이어진다. 현재까지 많은 연구와 노력에도 불구하고 비만과 당뇨 치료법들은 한계점은 가지고 있다. 비교적 최근에 도입된 대사 수술은 위장관계 수술을 통하여 체중을 감소시키고 나아가 당대사를 호전시키고자 하는 치료법이다. 현재까지 개발된 어떠한 약물치료에 비하여 대사 수술은 더 강력한 혈당 저하 및 체중감소 효과를 보이며, 이러한 효과는 10년이상 오래 유지됨이 밝혀졌다. 특히, 대사 수술은 당뇨 약제를 중단하여도 정상 혈당이 유지되는 당뇨 관해를 유도할 수도 있다고 알려졌다. 이러한 대사 수술의 기전을 연구함으로써 비만과 당뇨병을 치료할 수 있는 새로운 방향을 제시할 수 있을 것으로 기대된다. 이를 위하여, 본 연구에서 두 종류의 대사 수술 동물 모델을 연구하였다. 먼저 회장 전치술 렛트 모델을 통해서 대사 수술에서 원위 회장의 역할 및 수술 후 장의 적응 변화를 살펴보았다. 회장 전치술은 후위부 회장의 일부를 수술적으로 십이지장 다음으로 위치시켜 대사 수술에서 말단부 회장의 역할을 연구하고자 하는 수술법이다. 회장전치술을 고지방사료 섭취로 비만을 유도한 식이 유발 비만 렛트에서 시행하여 대사적인 효과를 살펴보았다. 회장전치술은 혈당을 감소시켰는데, 특히 식후 인슐린과 GLP-1 분비 증가를 통하여 이를 일으키는 것으로 생각되었다. 또한 이러한 변화는 췌장 베타세포의 증가와 동반되었다. 다음으로 전치된 회장에서의 유전자 발현 변화를 분석하였다. 회장 전치술 후에 1주, 4주가 지나고 회장 조직을 채취하고 마이크로어레이 분석을 시행하여 유전자 발현을 살펴보았다. 흥미롭게도, 1주와 4주의 유전자 발현사이에 큰 차이가 있어 회장 전치술 후 초기에 회장에서 매우 역동적인 적응 변화가 일어남을 알 수 있었다. 추가적인 유전자 경로 분석 및 네트워크 분석을 통하여 첫 1주에는 구조적인 적응이, 4주에는 대사적인 적응과 면역학적인 적응이 주된 반응임을 알 수 있었다. 다음으로, 당대사에 중요한 간, 지방, 근육 조직에서의 변화를 살펴보기 위하여 마우스에서 위소매절제술을 시행하여 연구하였다. 식이 유도 비만 마우스에서 위소매절제술을 시행한 뒤 간, 지방, 근육 조직을 채취하여 RNA 시퀀싱 분석을 통해 유전자 발현의 차이를 비교하였다. 또한 이를 sham 수술 대조군 뿐만 아니라 sham 수술 후 위소매절제술 수술과 동일한 식이를 섭취하도록 한 pair-fed 그룹과도 비교하여 위소매절제술의 효과에서 체중 감소 독립적인 부분을 확인하고자 하였다. 이러한 실험을 통하여 위소매절제술이 간, 지방, 근육에 모두 유전자 발현 변화를 유도하는데, 특히 이러한 차이는 단순한 식이 제한을 통해 체중 감소를 유도한 pair-fed 마우스에서 대조군과 비교한 차이보다 훨씬 크게 나타나서 위소매절제술의 간, 지방, 근육에 대한 효과 중 체중감소 독립적인 기전에 중요함을 알 수 있었다. 또한 면역 반응과 관련된 유전자들이 세가지 조직에서 모두 공통적으로 변화되어 있었다. 본 연구를 통해서 장에서는 수술 후 다이나믹한 적응 변화가 일어남을 알 수 있었고 말초에서 당을 대사하는 간, 지방, 근육 조직에서는 체중감소 독립적인 유전자 발현 변화가 크게 일어남을 알 수 있었다. 이러한 변화 중 면역 반응과 연관된 유전자 발현 변화가 모두 공통적으로 나타나서 대사 수술 후 면역 반응의 변화가 중요한 역할을 할 가능성을 시사하였다.Obesity increases the risk of metabolic, cardiovascular, musculoskeletal diseases and even some types of cancer. Among various complications of obesity, type 2 diabetes mellitus (T2DM) is the prototype of metabolic complications. Obesity is the primary contributor to the development of T2DM. Despite of tremendous efforts to bring better treatment for obesity and diabetes, there are still significant unmet needs. Metabolic surgery is a surgical treatment to reduce body weight and improve glucose metabolism. Unlike other pharmacologic treatment for obesity and diabetes, metabolic surgery demonstrated superior clinical efficacy and longer durability. Furthermore, metabolic surgery can induce remission of T2DM which is defined as achieving normoglycemia without any anti-diabetic medication. By unraveling the mechanism of metabolic surgery to improve metabolic homeostasis, we can develop new insights into glucose metabolism and better treatments. To elucidate the molecular mechanism of metabolic surgery, two rodent models of metabolic surgery were used. First, ileal transposition (IT) is an experimental surgery which translocate a segment of distal ileum into proximal jejunum. The metabolic effects of IT were investigated in diet-induced obese rats. IT improved glucose tolerance by augmenting postprandial insulin and GLP-1 secretion. IT also increased pancreatic β-cell mass which was thought to be associated with increased GLP-1 secretion. However, IT did not have significant effect on body weight and insulin sensitivity. On histologic examination, the transposed ileum showed hypertrophic change and increased enteroendocrine cell density positively stained for GLP-1 and GLP-1/GIP. Then, an unbiased transcriptomic analysis was applied on the transposed ileum to investigate the adaptive process of the intestine after IT. Using microarray analysis of the transposed ileum at week-1 and week-4 after surgery, I could demonstrate that the transposed ileum undergoes a dynamic adaptation process after IT. Functional enrichment analysis and network analysis further characterized the major components of each stage, which were structural adaptation at week 1 and metabolic and immune adaptations at week 4. Secondly, a mouse model of vertical sleeve gastrectomy (VSG) was constructed to investigate the effects of VSG in glucose metabolizing tissues including liver, fat and muscle. Vertical sleeve gastrectomy removes lateral 80% of the stomach and leaves only tube like remnant of the stomach. I examined gene expression profiles of liver, fat and muscle in VSG mice compared with sham and sham pair-fed (sham-PF) mice using RNA sequencing analysis at 8 weeks after surgery. VSG improved glucose tolerance significantly than sham, but only marginally than sham-PF. Unexpectedly, gene expression profiles of liver, fat, and muscle tissues were significantly different between VSG and sham-PF, which was more distinct than the comparison between sham and sham-PF, suggesting the weight-loss independent effects on gene expression of peripheral tissues are more robust than weight-loss dependent effects. In addition, immune response-related gene ontology and pathways were consistently un-regulated in the three organs. In further analysis, the immune response in adipose tissue was characterized as up-regulation of M2 phenotype macrophage and B lymphocytes. In summary, IT in rats and VSG in mice could demonstrate the metabolic improvements after metabolic surgery. The intestine undergoes dynamic adaptation process after surgery which can have implications in metabolic homeostasis with structural and functional changes. The glucose metabolizing peripheral tissues, liver, fat and muscle, showed both weight-loss dependent and independent changes in gene expression. The immune response was the common pathway enriched in the three organs after metabolic surgery.General Introduction 1 Chapter 1: Metabolic effects of ileal transposition and the adaptive process of the transposed distal ileum Background and objectives of chapter 1 27 Material and Methods 29 Results 39 Discussion of chapter 1 70 Chapter 2: Effects of vertical sleeve gastrectomy on the gene expression of liver, fat and muscle and their implications in glucose homeostasis Background and objectives of chapter 2 76 Material and Methods 78 Results 85 Discussion of chapter 2 124 Discussion 127 Acknowledgement 140 References 141 Abstract in Korean 159Docto

Genome-Wide Decoding of mRNP and miRNA Maps

The limited number of primary transcripts in the genome has promoted interest in the possibility that much of the complexity in the regulation of gene expression may be determined by RNA regulation controlled by RNA-binding proteins (RNABPs) and/or microRNAs (miRNAs). However, applying biochemical methods to understand such interactions in living tissues is major challenge. Here we developed a genome-wide means of mapping messenger ribonucleoprotein (mRNP) sites in vivo, by high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). HITS-CLIP analysis of the neuron-specific splicing factor Nova provides genome-wide maps of Nova-RNA interactions in vivo and leads to a new finding that Nova may regulate the processesing of some miRNAs. Furthermore, HITS-CLIP analysis is extended to the problem of identifying miRNA targets, for which prediction is a major challenge since miRNA activity requires base pairing through only 6-8 “seed†nucleotides. By generating crosslinking of native Argonaute (Ago) protein-RNA complexes in mouse brain, Ago HITS-CLIP produced two simultaneous datasets—Ago-miRNA and Ago-mRNA binding sites—that were combined with bioinformatic analysis to identify miRNA-target mRNA interaction sites. We validated genome-wide interaction maps for miR-124, and generated additional maps for the 20 most abundant miRNAs present in P13 mouse brain. We also found that the relatively large number of Ago proteins bind in coding sequence, as well as introns, suggesting unexplored functions for miRNAs. Not all Ago mRNA clusters correspond to known seed sequence, leading to the discovery of putative new rules for miRNA-mRNA interactions. HITS-CLIP provides a general plaform to identify functional mRNP and miRNA binding sites in vivo and a solution to determining precise sequences for targeting clinically relevant sites of RNA regulation. In addition, overlaying mRNP maps with miRNA maps will be informative for the understanding of RNA regulations and complexity

Statin-induced myopathic changes in primary human muscle cells and reversal by a prostaglandin F2 alpha analogue

Statin-related muscle side effects are a constant healthcare problem since patient compliance is dependent on side effects. Statins reduce plasma cholesterol levels and can prevent secondary cardiovascular diseases. Although statin-induced muscle damage has been studied, preventive or curative therapies are yet to be reported. We exposed primary human muscle cell populations (n = 22) to a lipophilic (simvastatin) and a hydrophilic (rosuvastatin) statin and analyzed their expressome. Data and pathway analyses included GOrilla, Reactome and DAVID. We measured mevalonate intracellularly and analyzed eicosanoid profiles secreted by human muscle cells. Functional assays included proliferation and differentiation quantification. More than 1800 transcripts and 900 proteins were differentially expressed after exposure to statins. Simvastatin had a stronger effect on the expressome than rosuvastatin, but both statins influenced cholesterol biosynthesis, fatty acid metabolism, eicosanoid synthesis, proliferation, and differentiation of human muscle cells. Cultured human muscle cells secreted ω-3 and ω-6 derived eicosanoids and prostaglandins. The ω-6 derived metabolites were found at higher levels secreted from simvastatin-treated primary human muscle cells. Eicosanoids rescued muscle cell differentiation. Our data suggest a new aspect on the role of skeletal muscle in cholesterol metabolism. For clinical practice, the addition of omega-n fatty acids might be suitable to prevent or treat statin-myopathy

Lung Cancer Genomic Signatures

Background:Lung cancer (LC) is the dominant cause of death by cancer in the world, being responsible for more than a million deaths annually. It is a highly lethal common tumor that is frequently diagnosed in advanced stages for which effective alternative therapeutics do not exist. In view of this, there is an urgent need to improve the diagnostic, prognostic, and therapeutic classification systems, currently based on clinicopathological criteria that do not adequately translate the enormous biologic complexity of this disease.Methods:The advent of the human genome sequencing project and the concurrent development of many genomic-based technologies have allowed scientists to explore the possibility of using expression profiles to identify homogenous tumor subtypes, new prognostic factors of human cancer, response to a particular treatment, etc. and thereby select the best possible therapies while decreasing the risk of toxicities for the patients. Therefore, it is becoming increasingly important to identify the complete catalog of genes that are altered in cancer and to discriminate tumors accurately on the basis of their genetic background.Results and Discussion:In this article, we present some of the works that has applied high-throughput technologies to LC research. In addition, we will give an overview of recent results in the field of LC genomics, with their effect on patient care, and discuss challenges and the potential future developments of this area

Genomic and proteomic profiling for cancer diagnosis in dogs

Global gene expression, whereby tumours are classified according to similar gene expression patterns or ‘signatures’ regardless of cell morphology or tissue characteristics, is being increasingly used in both the human and veterinary fields to assist in cancer diagnosis and prognosis. Many studies on canine tumours have focussed on RNA expression using techniques such as microarrays or next generation sequencing. However, proteomic studies combining two-dimensional polyacrylamide gel electrophoresis or two-dimensional differential gel electrophoresis with mass spectrometry have also provided a wealth of data on gene expression in tumour tissues. In addition, proteomics has been instrumental in the search for tumour biomarkers in blood and other body fluids

Implication of Spiritual Network Support System in Epigenomic Modulation and Health Trajectory

With challenges in understanding the multifactorial etiologies of disease and individual treatment effect heterogeneities over the past four decades, much has been acquired on how physical, chemical and social environments a ffect human health, predisposing certain subpopulations to adverse health outcomes, especially the socio-environmentally disadvantaged (SED). Current translational data on gene and adverse environment interaction have revealed how adverse gene-environment interaction, termed aberrant epigenomic modulation, translates into impaired gene expression via messenger ribonucleic acid (mRNA) dysregulation, reflecting abnormal protein synthesis and hence dysfunctional cellular differentiation and maturation. The environmental influence on gene expression observed in most literature includes physical, chemical, physicochemical and recently social environment. However, data are limited on spiritual or religious environment network support systems, which reflect human psychosocial conditions and gene interaction. With this limited information, we aimed to examine the available data on spiritual activities characterized by prayers and meditation for a possible explanation of the nexus between the spiritual network support system (SNSS) as a component of psychosocial conditions, implicated in social signal transduction, and the gene expression correlate. With the intent to incorporate SNSS in human psychosocial conditions, we assessed the available data on bereavement, loss of spouse, loneliness, social isolation, low socio-economic status (SES), chronic stress, low social status, social adversity (SA) and early life stress (ELS), as surrogates for spiritual support network connectome. Adverse human psychosocial conditions have the tendency for impaired gene expression through an up-regulated conserved transcriptional response to adversity (CTRA) gene expression via social signal transduction, involving the sympathetic nervous system (SNS), beta-adrenergic receptors, the hypothalamus-pituitary-adrenal (HPA) axis and the glucocorticoid response. This review specifically explored CTRA gene expression and the nuclear receptor subfamily 3 group C member 1 (NR3C1) gene, a glucocorticoid receptor gene, in response to stress and the impaired negative feedback, given allostatic overload as a result of prolonged and sustained stress and social isolation as well as the implied social interaction associated with religiosity. While more remains to be investigated on psychosocial and immune cell response and gene expression, current data on human models do implicate appropriate gene expression via the CTRA and NR3C1 gene in the SNSS as observed in meditation, yoga and thai-chi, implicated in malignant neoplasm remission. However, prospective epigenomic studies in this context are required in the disease causal pathway, prognosis and survival, as well as cautious optimism in the application of these findings in clinical and public health settings, due to unmeasured and potential confoundings implicated in these correlations