Faculty Bibliography

The goal of this talk is to introduce an integrated quantitative proteogenomic approach to comprehensively map proteomic information back to their encoding genes. We seek evidence from mass spectrometry-based large-scale proteomic data of patient populations in conjunction with patient-centric next-generation sequencing data and unbiased sequencing strategies to study breast cancer (BC) subtypes from a genomic context. We have obtained global and phosphoproteomic data with matching next generation sequencing data for 18 patient-derived xenografts (PDXs) representing the major clinical subtypes of BC. Our workflow starts with the creation of several protein sequence databases that serve as a template for mass spectrometry database identifications. These databases include 1) completely annotated reference protein sequences, 2) patient-specific databases that were created using next generation sequencing data, 3) isoform databases that contain all possible splicing combinations, and 4) amino acid sequence database resulting from a six-frame translation of the entire human reference and customized genomes. All mass spectrometry raw data are searched against the databases for obtaining identifications at the peptide level, and assembly of peptides for quantification using taxonomy-based label-free quantitation (LFQ) that can specifically quantify unique human peptide sequences found in PDXs. The peptides are then mapped to the human genome and visualized using a genome browser. Quantitative changes across PDXs are presented at the protein level or at the isoform level via peptide role-up to specific exons and visualized as a quantitative data track. By combining search results from these databases we obtain a comprehensive view of our PDXs. The complementary nature of the databases enable greater proteomic depth, i.e. databases with complete splicing combinations capture proteomic evidence when patient-specific databases fail due to possible erroneous RNA-seq reads. Similarly, 6-frame translated amino acid databases can capture potentially novel coding regions but are unable to detect splicing. Peptide maps are obtained for individual genes or specific protein isoforms covering both knowledge-driven and novel genomic annotation types. We compile peptides carrying variants, splice junctions, fusions, and new coding regions specific to each PDX or in common with a specific BC subtype. Majority of data is mapped back to the genome loci using unmodified peptides via global proteomics while phosphopeptides that contain variants and splicing are also mapped in a similar manner using phosphoproteomic data. We have currently annotated 455 novel proteogenomic hits covering many examples outlined above for genes related to breast cancer and show how these can be specifically identified and in some instances differentially quantified in the PDX models

Bullying is an intentional act that can wreak havoc in the life of an individual. With more than 1 billion users, YouTube^TM is a powerful medium for disseminating information. The purpose of this study was to describe the extent to which content related to bullying is present on YouTube^TM with respect to source, content, number of views, length, and year uploaded. Collectively, the videos in this sample were viewed more than half a billion times. The source of the most widely viewed videos was consumers, and none of the most widely viewed videos was posted by a governmental agency or a professional organization. The most common content in the videos was describing or depicting violence (n = 89). Over one-half addressed getting help (n = 56). Suicide was mentioned in 38 of the videos. Additional investment by professional agencies is warranted to improve understanding about ways to increase the dissemination of positive messages about bullying prevention, and about helping adolescents who are bullied on social media

Consistency, quality, and duration of sleep are important determinants of health. We describe sleep patterns among demographically defined subgroups from the Youth Risk Behavior Surveillance System reported in 4 successive biennial representative samples of American high school students (2007 to 2013). Across the 4 waves of data collection, 6.2% to 7.7% of females and 8.0% to 9.4% of males reported obtaining 9 or more hours of sleep. Insufficient duration of sleep is pervasive among American high school students. Despite substantive public health implications, intervention research on this topic has received little attention.

Adolescents are particularly vulnerable to engaging in poor skin-protection behaviors. The objective of this study was to examine use of sunscreen and indoor tanning devices among a nationally representative sample of high school students during a 10-year period (2001-2011) using data from the Youth Risk Behavior Surveillance System. The percentage of youth who reported using sunscreen declined from 67.7% in 2001 to 56.1% in 2011. The prevalence of using indoor tanning devices was highest among white females: 37.4% in 2009 and 29.3% in 2011. These findings indicate the need for prevention efforts aimed at adolescents to reduce risks for skin cancer.

Protein abundance and phosphorylation convey important information about pathway activity and molecular pathophysiology in diseases including cancer, providing biological insight, informing drug and diagnostic development, and guiding therapeutic intervention. Analyzed tissues are usually collected without tight regulation or documentation of ischemic time. To evaluate the impact of ischemia, we collected human ovarian tumor and breast cancer xenograft tissue without vascular interruption and performed quantitative proteomics and phosphoproteomics after defined ischemic intervals. While the global expressed proteome and most of the >25,000 quantified phosphosites were unchanged after 60 minutes, rapid phosphorylation changes were observed in up to 24% of the phosphoproteome, representing activation of critical cancer pathways related to stress response, transcriptional regulation and cell death. Both pan-tumor and tissue-specific changes were observed. The demonstrated impact of pre-analytical tissue ischemia on tumor biology mandates caution in interpreting stress-pathway activation in such samples, and motivates reexamination of collection protocols for phosphoprotein analysis.

The toxic subcellular accumulation of lipids predisposes several human metabolic syndromes, including obesity, type 2 diabetes, and some forms of neurodegeneration. To identify pathways that prevent lipid-induced cell death, we performed a genome-wide fatty acid sensitivity screen in Saccharomyces cerevisiae. We identified 167 yeast mutants as sensitive to 0.5 mm palmitoleate, 45% of which define pathways that were conserved in humans. 63 lesions also impacted the status of the lipid droplet; however, this was not correlated to the degree of fatty acid sensitivity. The most liposensitive yeast strain arose due to deletion of the "ARE2 required for viability" (ARV1) gene, encoding an evolutionarily conserved, potential lipid transporter that localizes to the endoplasmic reticulum membrane. Down-regulation of mammalian ARV1 in MIN6 pancreatic beta-cells or HEK293 cells resulted in decreased neutral lipid synthesis, increased fatty acid sensitivity, and lipoapoptosis. Conversely, elevated expression of human ARV1 in HEK293 cells or mouse liver significantly increased triglyceride mass and lipid droplet number. The ARV1-induced hepatic triglyceride accumulation was accompanied by up-regulation of DGAT1, a triglyceride synthesis gene, and the fatty acid transporter, CD36. Furthermore, ARV1 was identified as a transcriptional of the protein peroxisome proliferator-activated receptor alpha (PPARalpha), a key regulator of lipid homeostasis whose transcriptional targets include DGAT1 and CD36. These results implicate ARV1 as a protective factor in lipotoxic diseases due to modulation of fatty acid metabolism. In conclusion, a lipotoxicity-based genetic screen in a model microorganism has identified 75 human genes that may play key roles in neutral lipid metabolism and disease.

OBJECTIVE: To apply discovery-based computational methods to nationally representative data from the Centers for Disease Control and Preventions' Youth Risk Behavior Surveillance System to better understand and visualize the behavioral factors associated with gun possession among adolescent youth. RESULTS: Our study uncovered the multidimensional nature of gun possession across nearly five million unique data points over a ten year period (2001-2011). Specifically, we automated odds ratio calculations for 55 risk behaviors to assemble a comprehensive table of associations for every behavior combination. Downstream analyses included the hierarchical clustering of risk behaviors based on their association "fingerprint" to 1) visualize and assess which behaviors frequently co-occur and 2) evaluate which risk behaviors are consistently found to be associated with gun possession. From these analyses, we identified more than 40 behavioral factors, including heroin use, using snuff on school property, having been injured in a fight, and having been a victim of sexual violence, that have and continue to be strongly associated with gun possession. Additionally, we identified six behavioral clusters based on association similarities: 1) physical activity and nutrition; 2) disordered eating, suicide and sexual violence; 3) weapon carrying and physical safety; 4) alcohol, marijuana and cigarette use; 5) drug use on school property and 6) overall drug use. CONCLUSIONS: Use of computational methodologies identified multiple risk behaviors, beyond more commonly discussed indicators of poor mental health, that are associated with gun possession among youth. Implications for prevention efforts and future interdisciplinary work applying computational methods to behavioral science data are described.