Application of Proteomics to Human Hypertrophic Cardiomyopathy

This work describes a preliminary study to evaluate the use of proteomics in the study of human hypertrophic cardiomyopathy. Both gel and mass spectrometry techniques were used for the identification and analysis of myocardial proteins in whole tissue lysate. Qualitative and quantitative proteomic methods were used to understand disease mechanisms and identify and validate novel biomarkers. Disease caused by mutations in beta myosin heavy chain, MYH7 and myosin binding protein-C, MYBPC3 were studied alongside patients where no genetic variant could be identified. Left ventricular septal myectomy samples showed changes in protein expression compared with control tissue. Novel mutations in MYH7 were confirmed by identification of mutant peptide sequences. Disease mechanisms were investigated by studying interactions between up- and down-regulated proteins involved in various pathways. Enriched protein groups included those involved in cytoskeletal protein binding and energy production. Novel findings included the identification of carbonic anhydrase III in cardiomyocytes. A targeted and multiplexed MRM-MS assay was developed to validate potential biomarkers in tissue and correlated with clinical phenotype. The assay was further applied to screen these biomarkers in urine. Novel findings included increased expression of lumican, a small leucine-rich proteoglycan that controls the assembly of collagen fibres in the extracellular matrix. Lumican concentration was highest in a sub-group of patients with evidence of scarring on cardiac magnetic resonance imaging, making it a potential marker of progressive disease. This is the first comprehensive global proteomic study of human hypertrophic cardiomyopathy. It has identified differences in the expression of several proteins in the myocardium not described previously, but highly relevant to pathophysiology of this disease. A targeted proteomic translational assay, capable of quantitating 35 peptides in less than 10 minutes has been developed

Frameshift mutations at the C-terminus of HIST1H1E result in a specific DNA hypomethylation signature

BACKGROUND: We previously associated HIST1H1E mutations causing Rahman syndrome with a specific genome-wide methylation pattern. RESULTS: Methylome analysis from peripheral blood samples of six affected subjects led us to identify a specific hypomethylated profile. This "episignature" was enriched for genes involved in neuronal system development and function. A computational classifier yielded full sensitivity and specificity in detecting subjects with Rahman syndrome. Applying this model to a cohort of undiagnosed probands allowed us to reach diagnosis in one subject. CONCLUSIONS: We demonstrate an epigenetic signature in subjects with Rahman syndrome that can be used to reach molecular diagnosis

Atherogenesis

This monograph will bring out the state-of-the-art advances in the dynamics of cholesterol transport and will address several important issues that pertain to oxidative stress and inflammation. The book is divided into three major sections. The book will offer insights into the roles of specific cytokines, inflammation, and oxidative stress in atherosclerosis and is intended for new researchers who are curious about atherosclerosis as well as for established senior researchers and clinicians who would be interested in novel findings that may link various aspects of the disease

NASH and Systemic Complications

Nonalcoholic fatty liver disease (NAFLD) is known as the hepatic manifestation of the metabolic syndrome, and while most patients develop simple steatosis, up to one-third can develop nonalcoholic steatohepatitis (NASH). NASH is a chronic inflammatory condition of the liver that can further progress to fibrosis and cirrhosis, which may eventually lead to liver failure and death. While we have increased our mechanistic knowledge regarding the pathogenesis of NASH within the last decade, treatment options are still limited and liver biopsies have remained the gold standard for diagnosis. To achieve major clinical breakthroughs for NASH patients, it is not sufficient to use a single animal model, since each model has specific limitations. Furthermore, we should rely more on alternative models such as organ-on-a-chip, which will enable us to explore unknown aspects of disease pathogenesis much faster and serve as clinically relevant surrogates for murine models. Another important direction for the improvement of patient health is to pay more attention to extrahepatic, organ-specific and systemic effects, which are associated with NASH. The articles in this Special Issue include an up-to-date overview of the rapidly developing technologies, novel targets for intervention and insights in the field in NASH. Additionally, these articles describe the major challenges in the field, strategies to overcome them and suggestions for future directions. To improve patient’s outcome, clinicians, as well as scientists with biomedical, nutrition, physics and mathematics backgrounds, should join forces. Although challenges remain, the future of the field seems promising as these novel technologies and developments are expected to lead to progress in NASH

Methods and tools for mining the transcriptomic landscape of human tissue and disease

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2012.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from PDF version of thesis.Includes bibliographical references (p. 343-356).Although there are a variety of high-throughput technologies used to perform biological experiments, DNA microarrays have become a standard tool in the modern biologist's arsenal. Microarray experiments provide measurements of thousands of genes simultaneously, and offer a snapshot view of transcriptomic activity. With the rapid growth of public availability of transcriptomic data, there is increasing recognition that large sets of such data can be mined to better understand disease states and mechanisms. Unfortunately, several challenges arise when attempting to perform such large-scale analyses. For instance, public repositories to which the data is being submitted to were designed around the simple task of storage rather than that of data mining. As such, the seemingly simple task of obtaining all data relating to a particular disease becomes an arduous task. Furthermore, prior gene expression analyses, both large and small, have been dichotomous in nature, in which phenotypes are compared using clearly defined controls. Such approaches may require arbitrary decisions about what are considered "normal" phenotypes, and what each phenotype should be compared to. Addressing these issues, we introduce methods for creating a large curated gene expression database geared towards data mining, and explore methods for efficiently expanding this database using active learning. Leveraging our curated expression database, we adopt a holistic approach in which we characterize phenotypes in the context of a myriad of tissues and diseases. We introduce scalable methods that associate expression patterns to phenotypes in order to assign phenotype labels to new expression samples and to select phenotypically meaningful gene signatures. By using a nonparametric statistical approach, we identify signatures that are more precise than those from existing approaches and accurately reveal biological processes that are hidden in case vs. control studies. We conclude the work by exploring the applicability of the heterogeneous expression database in analyzing clinical drugs for the purpose of drug repurposing.by Patrick Raphael Schmid.Ph.D

Diagnostic Significance of Exosomal miRNAs in the Plasma of Breast Cancer Patients

Poster Session AbstractsBackground and Aims: Emerging evidence that microRNAs (miRNAs) play an important role in cancer development has opened up new opportunities for cancer diagnosis. Recent studies demonstrated that released exosomes which contain a subset of both cellular mRNA and miRNA could be a useful source of biomarkers for cancer detection. Here, we aim to develop a novel biomarker for breast cancer diagnosis using exosomal miRNAs in plasma. Methods: We have developed a rapid and novel isolation protocol to enrich tumor-associated exosomes from plasma samples by capturing tumor specific surface markers containing exosomes. After enrichment, we performed miRNA profiling on four sample sets; (1) Ep-CAM marker enriched plasma exosomes of breast cancer patients; (2) breast tumors of the same patients; (3) adjacent non-cancerous tissues of the same patients; (4) Ep-CAM marker enriched plasma exosomes of normal control subjects. Profiling is performed using PCR-based array with human microRNA panels that contain more than 700 miRNAs. Results: Our profiling data showed that 15 miRNAs are concordantly up-regulated and 13 miRNAs are concordantly down-regulated in both plasma exosomes and corresponding tumors. These account for 25% (up-regulation) and 15% (down-regulation) of all miRNAs detectable in plasma exosomes. Our findings demonstrate that miRNA profile in EpCAM-enriched plasma exosomes from breast cancer patients exhibit certain similar pattern to that in the corresponding tumors. Based on our profiling results, plasma signatures that differentiated breast cancer from control are generated and some of the well-known breast cancer related miRNAs such as miR-10b, miR-21, miR-155 and miR-145 are included in our panel list. The putative miRNA biomarkers are validated on plasma samples from an independent cohort from more than 100 cancer patients. Further validation of the selected markers is likely to offer an accurate, noninvasive and specific diagnostic assay for breast cancer. Conclusions: These results suggest that exosomal miRNAs in plasma may be a novel biomarker for breast cancer diagnosis.link_to_OA_fulltex

Atherosclerosis: Methods and Protocols

This volume provides detailed, up-to-date methods used in research on Atherosclerosis. Chapters guide readers through an overview of the pathogenesis of atherosclerosis and model systems together with in vitro, ex vivo, in vivo and emerging methods in atherosclerosis research. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Atherosclerosis: Methods and Protocols serves as an invaluable resource for those engaging in research on atherosclerosis and cardiovascular disease, as well as for researchers who are new to t