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Objective:Peripheral artery disease (PAD) is an atherothrombotic disease of the lower limbs with substantial morbidity and mortality. We used next-generation sequencing to identify genome-wide expression signatures associated with prevalent PAD and its outcomes.Approach and Results:We performed whole blood RNA sequencing among patients with severe symptomatic PAD undergoing lower extremity revascularization and controls. Dysregulated pathways and blood transcriptional modules were identified by comparing patients with PAD (n=42) to age- and sex-matched controls (N=29). The identified signature was compared in patients with PAD before LER with or without incident major adverse cardiac or limb events (MACLE). A novel microRNA associated with prevalent PAD and incident MACLE was then evaluated in a mouse hindlimb ischemia model. One hundred twenty-seven transcripts were differentially expressed (77 upregulated and 50 downregulated; adjustedP0.5) and analyzed using weighted gene co-expression network analysis. Weighted gene co-expression network analysis revealed blood modules enriched for immune activation, secretory granules, and coagulation in patients with PAD. Of these 127 differentially expressed transcripts, 40 were significantly associated with MACLE (log-rank false discovery rate Conclusions:A whole blood transcript signature identified patients with symptomatic PAD and PAD patients at increased risk of MACLE. A previously uncharacterized transcript microRNA-4477b was overexpressed in prevalent PAD, incident MACLE, and in a mouse hindlimb ischemia model. Our novel transcriptomic signature provides insight into potential mechanisms of patients with severe symptomatic PAD.

Background Unsupervised clustering is a common and exceptionally useful tool for large biological datasets. However, clustering requires upfront algorithm and hyperparameter selection, which can introduce bias into the final clustering labels. It is therefore advisable to obtain a range of clustering results from multiple models and hyperparameters, which can be cumbersome and slow. Results We present hypercluster, a python package and SnakeMake pipeline for flexible and parallelized clustering evaluation and selection. Users can efficiently evaluate a huge range of clustering results from multiple models and hyperparameters to identify an optimal model. Conclusions Hypercluster improves ease of use, robustness and reproducibility for unsupervised clustering application for high throughput biology. Hypercluster is available on pip and bioconda; installation, documentation and example workflows can be found at: https://github.com/ruggleslab/hypercluster.

Aberrant phospho-signaling is a hallmark of cancer. We investigated kinase-substrate regulation of 33,239 phosphorylation sites (phosphosites) in 77 breast tumors and 24 breast cancer xenografts. Our search discovered 2134 quantitatively correlated kinase-phosphosite pairs, enriching for and extending experimental or binding-motif predictions. Among the 91 kinases with auto-phosphorylation, elevated EGFR, ERBB2, PRKG1, and WNK1 phosphosignaling were enriched in basal, HER2-E, Luminal A, and Luminal B breast cancers, respectively, revealing subtype-specific regulation. CDKs, MAPKs, and ataxia-telangiectasia proteins were dominant, master regulators of substrate-phosphorylation, whose activities are not captured by genomic evidence. We unveiled phospho-signaling and druggable targets from 113 kinase-substrate pairs and cascades downstream of kinases, including AKT1, BRAF and EGFR. We further identified kinase-substrate-pairs associated with clinical or immune signatures and experimentally validated activated phosphosites of ERBB2, EIF4EBP1, and EGFR. Overall, kinase-substrate regulation revealed by the largest unbiased global phosphorylation data to date connects driver events to their signaling effects.

Unbiased assays such as shotgun proteomics and RNA-seq provide high-resolution molecular characterization of tumors. These assays measure molecules with highly varied distributions, making interpretation and hypothesis testing challenging. Samples with the most extreme measurements for a molecule can reveal the most interesting biological insights, yet are often excluded from analysis. Furthermore, rare disease subtypes are, by definition, underrepresented in cancer cohorts. To provide a strategy for identifying molecules aberrantly enriched in small sample cohorts, we present BlackSheep--a package for non-parametric description and differential analysis of genome-wide data, available at https://github.com/ruggleslab/blackSheep. BlackSheep is a complementary tool to other differential expression analysis methods that may be underpowered when analyzing small subgroups in a larger cohort.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of Coronavirus Disease 2019 (COVID-19). There is a dire need for novel effective antivirals to treat COVID-19, as the only approved direct-acting antiviral to date is remdesivir, targeting the viral polymerase complex. A potential alternate target in the viral life cycle is the main SARS-CoV-2 protease 3CLpro (Mpro). The drug candidate PF-00835231 is the active compound of the first anti-3CLpro regimen in clinical trials. Here, we perform a comparative analysis of PF-00835231, the pre-clinical 3CLpro inhibitor GC-376, and the polymerase inhibitor remdesivir, in alveolar basal epithelial cells modified to express ACE2 (A549+ACE2 cells). We find PF-00835231 with at least similar or higher potency than remdesivir or GC-376. A time-of-drug-addition approach delineates the timing of early SARS-CoV-2 life cycle steps in A549+ACE2 cells and validates PF-00835231's early time of action. In a model of the human polarized airway epithelium, both PF-00835231 and remdesivir potently inhibit SARS-CoV-2 at low micromolar concentrations. Finally, we show that the efflux transporter P-glycoprotein, which was previously suggested to diminish PF-00835231's efficacy based on experiments in monkey kidney Vero E6 cells, does not negatively impact PF-00835231 efficacy in either A549+ACE2 cells or human polarized airway epithelial cultures. Thus, our study provides in vitro evidence for the potential of PF-00835231 as an effective SARS-CoV-2 antiviral and addresses concerns that emerged based on prior studies in non-human in vitro models.Importance:The arsenal of SARS-CoV-2 specific antiviral drugs is extremely limited. Only one direct-acting antiviral drug is currently approved, the viral polymerase inhibitor remdesivir, and it has limited efficacy. Thus, there is a substantial need to develop additional antiviral compounds with minimal side effects and alternate viral targets. One such alternate target is its main protease, 3CLpro (Mpro), an essential component of the SARS-CoV-2 life cycle processing the viral polyprotein into the components of the viral polymerase complex. In this study, we characterize a novel antiviral drug, PF-00835231, which is the active component of the first-in-class 3CLpro-targeting regimen in clinical trials. Using 3D in vitro models of the human airway epithelium, we demonstrate the antiviral potential of PF-00835231 for inhibition of SARS-CoV-2.

Advances in mass-spectrometry have generated increasingly large-scale proteomics datasets containing tens of thousands of phosphorylation sites (phosphosites) that require prioritization. We develop a bioinformatics tool called HotPho and systematically discover 3D co-clustering of phosphosites and cancer mutations on protein structures. HotPho identifies 474 such hybrid clusters containing 1255 co-clustering phosphosites, including RET p.S904/Y928, the conserved HRAS/KRAS p.Y96, and IDH1 p.Y139/IDH2 p.Y179 that are adjacent to recurrent mutations on protein structures not found by linear proximity approaches. Hybrid clusters, enriched in histone and kinase domains, frequently include expression-associated mutations experimentally shown as activating and conferring genetic dependency. Approximately 300 co-clustering phosphosites are verified in patient samples of 5 cancer types or previously implicated in cancer, including CTNNB1 p.S29/Y30, EGFR p.S720, MAPK1 p.S142, and PTPN12 p.S275. In summary, systematic 3D clustering analysis highlights nearly 3,000 likely functional mutations and over 1000 cancer phosphosites for downstream investigation and evaluation of potential clinical relevance., Dysregulated phosphorylation is well-known in cancers, but it has largely been studied in isolation from mutations. Here the authors introduce HotPho, a tool that can discover spatial interactions between phosphosites and mutations, which are associated with activating mutation and genetic dependencies in cancer.

Atherosclerosis is a disease of chronic inflammation. We investigated the roles of the cytokines IL-4 and IL-13, the classical activators of STAT6, in the resolution of atherosclerosis inflammation. Using Il4-/-Il13-/- mice, resolution was impaired, and in control mice, in both progressing and resolving plaques, levels of IL-4 were stably low and IL-13 was undetectable. This suggested that IL-4 is required for atherosclerosis resolution, but collaborates with other factors. We had observed increased Wnt signaling in macrophages in resolving plaques, and human genetic data from others showed that a loss-of-function Wnt mutation was associated with premature atherosclerosis. We now find an inverse association between activation of Wnt signaling and disease severity in mice and humans. Wnt enhanced the expression of inflammation resolving factors after treatment with plaque-relevant low concentrations of IL-4. Mechanistically, activation of the Wnt pathway following lipid lowering potentiates IL-4 responsiveness in macrophages via a PGE2/STAT3 axis.

Within the last decade, numerous studies have demonstrated changes in the gut microbiome associated with specific autoimmune diseases. Due to differences in study design, data quality control, analysis and statistical methods, many results of these studies are inconsistent and incomparable. To better understand the relationship between the intestinal microbiome and autoimmunity, we have completed a comprehensive re-analysis of 42 studies focusing on the gut microbiome in twelve autoimmune diseases to identify a microbial signature predictive of multiple sclerosis (MS), inflammatory bowel disease (IBD), rheumatoid arthritis (RA) and general autoimmune disease using both 16S rRNA sequencing data and shotgun metagenomics data. To do this, we used four machine learning algorithms, random forest, eXtreme Gradient Boosting (XGBoost), ridge regression and support vector machine with radial kernel and recursive feature elimination to rank disease predictive taxa comparing disease vs. healthy participants and pairwise comparisons of each disease. Comparing the performance of these models, we found XGBoost and random forest, tree-based methods capable of handling sparse multidimensional data, to consistently produce the best results. Through this modeling, we identified a number of taxa consistently identified as dysregulated in a general autoimmune disease model including Odoribacter, Lachnospiraceae Clostridium and Mogibacteriaceae implicating all as potential factors connecting the gut microbiome and to autoimmune response. Further, we computed pairwise comparison models to identify disease specific taxa signatures highlighting a role for Peptostreptococcaceae and Ruminococcaceae Gemmiger in IBD and Akkermansia, Butyricicoccus and Mogibacteriaceae in MS. We then connected a subset of these taxa with potential metabolic alterations based on metagenomic/metabolomic correlation analysis, identifying 250 metabolites associated with autoimmunity-predictive taxa.

Over-accumulation of oxalate in humans may lead to nephrolithiasis and nephrocalcinosis. Humans lack endogenous oxalate degradation pathways (ODP), but intestinal microbes can degrade oxalate using multiple ODPs and protect against its absorption. The exact oxalate-degrading taxa in the human microbiota and their ODP have not been described. We leverage multi-omics data (>3000 samples from >1000 subjects) to show that the human microbiota primarily uses the type II ODP, rather than type I. Furthermore, among the diverse ODP-encoding microbes, an oxalate autotroph, Oxalobacter formigenes, dominates this function transcriptionally. Patients with inflammatory bowel disease (IBD) frequently suffer from disrupted oxalate homeostasis and calcium oxalate nephrolithiasis. We show that the enteric oxalate level is elevated in IBD patients, with highest levels in Crohn’s disease (CD) patients with both ileal and colonic involvement consistent with known nephrolithiasis risk. We show that the microbiota ODP expression is reduced in IBD patients, which may contribute to the disrupted oxalate homeostasis. The specific changes in ODP expression by several important taxa suggest that they play distinct roles in IBD-induced nephrolithiasis risk. Lastly, we colonize mice that are maintained in the gnotobiotic facility with O. formigenes, using either a laboratory isolate or an isolate we cultured from human stools, and observed a significant reduction in host fecal and urine oxalate levels, supporting our in silico prediction of the importance of the microbiome, particularly O. formigenes in host oxalate homeostasis.

Background Among adolescents, the pathway from being offered drugs to engaging in drug use has been established. The prevalence of drug transactions specifically in schools is less understood. The purpose of this study was to identify the prevalence of adolescents who have reported drug transaction experiences (being offered, sold, or given an illegal drug) on school property and subsequently identify behavioral correlates associated with these experiences. Methods This study analyzed YRBS data from 2001 to 2015 (N = 117,815). We used 95% confidence intervals to evaluate differences in prevalence across years and demographic subgroups. Odds ratios determined the likelihood of engaging in risk behaviors among those youth also experiencing drug transactions on school property. Results Every survey year, at least 1 in 5 youth reported having drug transaction experiences at school. Boys consistently reported significantly higher rates of drug transaction experiences in comparison to girls, with Hispanic boys representing the highest rates. Youth reporting these experiences with drug transactions were also more likely to carry a weapon to school and to be bullied at school. Conclusions Implications for the reduction of drug transactions in high schools are discussed and recommendations for prevention efforts addressing youth engagement in multiple risk behaviors are identified.

ObjectiveHydroxychloroquine (HCQ) is a mainstay of therapy in the treatment of SLE. The effect of HCQ on platelets and vascular health is uncertain. We investigated the relationship between HCQ use and dose with platelet activity, platelet transcriptomics and vascular health in patients with SLE.MethodsPlatelet aggregation, platelet mRNA expression and vascular health (sublingual capillary perfused boundary region (PBR), red blood cell filling (RBCF) and brachial artery reactivity testing) were analysed by HCQ use and dose.ResultsAmong 132 subjects with SLE (age: 39.7±12.9 years, 97% female), 108 were on HCQ. SLE disease activity was similar between subjects on and off HCQ. Platelet aggregation in response to multiple agonists was significantly lower in patients on HCQ. There were inverse relationships between HCQ dose and gene expression pathways of platelet activity. Gene expression of P-selectin (SELP) was inversely correlated with HCQ dose (r=−0.41, p=0.003), which was validated at the protein level. Subjects on HCQ had improved vascular function correlating with HCQ dose as measured by lower PBR (r=−0.52, p=0.007), higher RBCF (r=0.55, p=0.004) and greater brachial artery reactivity (r=0.43, p=0.056).ConclusionHCQ use was associated with decreased platelet activation and activation-related transcripts and improved vascular health in SLE.

Glioblastoma (GBM) is the most aggressive nervous system cancer. Understanding its molecular pathogenesis is crucial to improving diagnosis and treatment. Integrated analysis of genomic, proteomic, post-translational modification and metabolomic data on 99 treatment-naive GBMs provides insights to GBM biology. We identify key phosphorylation events (e.g., phosphorylated PTPN11 and PLCG1) as potential switches mediating oncogenic pathway activation, as well as potential targets for EGFR-, TP53-, and RB1-altered tumors. Immune subtypes with distinct immune cell types are discovered using bulk omics methodologies, validated by snRNA-seq, and correlated with specific expression and histone acetylation patterns. Histone H2B acetylation in classical-like and immune-low GBM is driven largely by BRDs, CREBBP, and EP300. Integrated metabolomic and proteomic data identify specific lipid distributions across subtypes and distinct global metabolic changes in IDH-mutated tumors. This work highlights biological relationships that could contribute to stratification of GBM patients for more effective treatment. Wang et al. perform integrated proteogenomic analysis of adult glioblastoma (GBM), including metabolomics, lipidomics, and single nuclei RNA-Seq, revealing insights into the immune landscape of GBM, cell-specific nature of EMT signatures, histone acetylation in classical GBM, and the existence of signaling hubs which could provide therapeutic vulnerabilities.

Introduction: Although a substantial body of research documents a relationship between traumatic stress in childhood and the initiation of substance use later in the life course, only limited research has examined potential linkages between adverse childhood experiences (ACEs) and the initiation of non-medical prescription opioid use and other opioid use behaviors. The present study contributes to this growing body of work by investigating the association of childhood trauma with early initiation of a series of opioid use behaviors.Methods: New York City young adults (n = 539) ages 18–29 who reported non-medical use of prescription opioids or heroin use in the past 30 days were recruited using Respondent-Driven Sampling in 2014–16. Ten ACEs were assessed via self-report with the ACE Questionnaire. Associations between number of ACEs and self-reported ages of initiating seven opioid use behaviors (e.g., non-medical prescription opioid use, heroin use, heroin injection) were estimated with multivariable logistic regression.Results: Eighty nine percent of participants reported at least one ACE, and 46% reported four or more ACEs, a well-supported threshold indicating elevated risk for negative health consequences. Every increase of one trauma was associated with a 12–23% increase in odds of early initiation across the seven opioid use behaviors. Findings also document that the mean age at initiation increased with increasing risk severity across the behaviors, contributing to evidence of a trajectory from opioid pill misuse to opioid injection.Discussion: Increasing number of childhood traumas was associated with increased odds of earlier initiation of multiple opioid misuse behaviors. In light of prior research linking earlier initiation of substance use with increased substance use severity, present findings suggest the importance of ACEs as individual-level determinants of increased opioid use severity. Efforts to prevent onset and escalation of opioid use among at-risk youth may benefit from trauma prevention programs and trauma-focused screening and treatment, as well as increased attention to ameliorating upstream socio-structural drivers of childhood trauma.

SUMMARY: Many gene products exhibit great structural heterogeneity because of an array of modifications. These modifications are not directly encoded in the genomic template but often affect the functionality of proteins. Protein glycosylation plays a vital role in proper protein functions. However, the analysis of glycoproteins has been challenging compared with other protein modifications, such as phosphorylation. Here, we perform an integrated proteomic and glycoproteomic analysis of 83 prospectively collected high-grade serous ovarian carcinoma (HGSC) and 23 non-tumor tissues. Integration of the expression data from global proteomics and glycoproteomics reveals tumor-specific glycosylation, uncovers different glycosylation associated with three tumor clusters, and identifies glycosylation enzymes that were correlated with the altered glycosylation. In addition to providing a valuable resource, these results provide insights into the potential roles of glycosylation in the pathogenesis of HGSC, with the possibility of distinguishing pathological outcomes of ovarian tumors from non-tumors, as well as classifying tumor clusters. IN BRIEF: Hu et al. provide an integrated proteomic and glycoproteomic characterization of high-grade serous ovarian carcinomas and relevant non-tumor tissues, which reveals tumor-specific glycosylation, uncovers different glycosylation associated with three tumor clusters, and identifies glycosylation enzymes correlated with glycosylation alterations.

Background Benzodiazepines are a widely prescribed psychoactive drug; in the U.S., both medical and nonmedical use of benzodiazepines has increased markedly in the past 15 years. Long-term use can lead to tolerance and dependence, and abrupt withdrawal can cause seizures or other life-threatening symptoms. Benzodiazepines are often used nonmedically in conjunction with other drugs, and with opioids in particular—a combination that can increase the risk for fatal and non-fatal overdose. This mixed-methods study examines nonmedical use of benzodiazepines among young adults in New York City and its relationship with opioid use. Methods For qualitative analysis, 46 90-minute semi-structured interviews were conducted with young adult opioid users (ages 18–32). Interviews were transcribed and coded for key themes. For quantitative analysis, 464 young adult opioid users (ages 18–29) were recruited using Respondent-Driven Sampling and completed structured interviews. Benzodiazepine use was assessed via a self-report questionnaire that included measures related to nonmedical benzodiazepine and opioid use. Results Participants reported using benzodiazepines nonmedically for a wide variety of reasons, including: to increase the high of other drugs; to lessen withdrawal symptoms; and to come down from other drugs. Benzodiazepines were described as readily available and cheap. There was a high prevalence (93%) of nonmedical benzodiazepine use among nonmedical opioid users, with 57% reporting regular nonmedical use. In bivariate analyses, drug-related risk behaviours such as polysubstance use, drug binging, heroin injection and overdose were strongly associated with regular nonmedical benzodiazepine use. In multivariate analysis, growing up in a middle-income household (earning between $51,000 and $100,000 annually), lifetime overdose experience, having ever used cocaine regularly, having ever been prescribed benzodiazepines, recent drug binging, and encouraging fellow drug users to use benzodiazepines to cope with opioid withdrawal were consistently strong predictors of regular nonmedical benzodiazepine use. Conclusion Nonmedical benzodiazepine use may be common among nonmedical opioid users due to its drug-related multi-functionality. Harm reduction messages should account for the multiple functions benzodiazepines serve in a drug-using context, and encourage drug users to tailor their endorsement of benzodiazepines to peers to include safer alternatives.

With combined technological advancements in high-throughput next-generation sequencing and deep mass spectrometry-based proteomics, proteogenomics, i.e. the integrative analysis of proteomic and genomic data, has emerged as a new research field. Early efforts in the field were focused on improving protein identification using sample-specific genomic and transcriptomic sequencing data. More recently, integrative analysis of quantitative measurements from genomic and proteomic studies have identified novel insights into gene expression regulation, cell signaling, and disease. Many methods and tools have been developed or adapted to enable an array of integrative proteogenomic approaches and in this article, we systematically classify published methods and tools into four major categories, (1) Sequence-centric proteogenomics; (2) Analysis of proteogenomic relationships; (3) Integrative modeling of proteogenomic data; and (4) Data sharing and visualization. We provide a comprehensive review of methods and available tools in each category and highlight their typical applications.

Over-accumulation of oxalate in humans may lead to nephrolithiasis and nephrocalcinosis. Humans lack endogenous oxalate degradation pathways (ODP), but intestinal microbiota can degrade oxalate and protect against its absorption. However, the particular microbes that actively degrade oxalatein vivoare ill-defined, which restricts our ability to disentangle the underlying taxonomic contributions. Here we leverage large-scale multi-omics data (>3000 samples from >1000 subjects) to show that the human microbiota in health harbors diverse ODP-encoding microbial species, but an oxalate autotroph-Oxalobacter formigenes-dominates this function transcriptionally. Patients with Inflammatory Bowel Disease (IBD) are at significantly increased risk for disrupted oxalate homeostasis and calcium-oxalate nephrolithiasis. Here, by analyzing multi-omics data from the iHMP-IBD study, we demonstrate that the oxalate degradation function conferred by the intestinal microbiota is severely impaired in IBD patients. In parallel, the enteric oxalate levels of IBD patients are significantly elevated and associated with intestinal disease severity, which is consistent with the clinically known nephrolithiasis risk. The specific changes in ODP expression by several important taxa suggest that they play different roles in the IBD-induced nephrolithiasis risk.

SummaryBrain metastasis is a significant cause of morbidity and mortality in multiple cancer types and represents an unmet clinical need. The mechanisms that mediate metastatic cancer growth in the brain parenchyma are largely unknown. Melanoma, which has the highest rate of brain metastasis among common cancer types, is an ideal model to study how cancer cells adapt to the brain parenchyma. We performed unbiased proteomics analysis of melanoma short-term cultures, a novel model for the study of brain metastasis. Intriguingly, we found that proteins implicated in neurodegenerative pathologies are differentially expressed in melanoma cells explanted from brain metastases compared to those derived from extracranial metastases. This raised the exciting hypothesis that molecular pathways implicated in neurodegenerative disorders are critical for metastatic adaptation to the brain.Here, we show that melanoma cells require amyloid beta (Aβ), a polypeptide heavily implicated in Alzheimer’s disease, for growth and survival in the brain parenchyma. Melanoma cells produce and secrete Aβ, which activates surrounding astrocytes to a pro-metastatic, anti-inflammatory phenotype. Furthermore, we show that pharmacological inhibition of Aβ decreases brain metastatic burden.Our results reveal a mechanistic connection between brain metastasis and Alzheimer’s disease – two previously unrelated pathologies, establish Aβ as a promising therapeutic target for brain metastasis, and demonstrate suppression of neuroinflammation as a critical feature of metastatic adaptation to the brain parenchyma.

Summary The relative contributions of genetic and environmental factors to variation in immune responses are poorly understood. Here, we performed a phenotypic analysis of immunological parameters in laboratory mice carrying susceptibility genes implicated in inflammatory bowel disease (IBD) (Nod2 and Atg16l1) upon exposure to environmental microbes. Mice were released into an outdoor enclosure (rewilded) and then profiled for immune responses in the blood and lymph nodes. Variations of immune cell populations were largely driven by the environment, whereas cytokine production elicited by microbial antigens was more affected by the genetic mutations. We identified transcriptional signatures in the lymph nodes associated with differences in T cell populations. Subnetworks associated with responses against Clostridium perfringens, Candida albicans, and Bacteroides vulgatus were also coupled with rewilding. Therefore, exposing laboratory mice with genetic mutations to a natural environment uncovers different contributions to variations in microbial responses and immune cell composition.

Myeloid cells are a vital component of innate immunity and comprise of monocytes, macrophages, dendritic cells and granulocytes. How myeloid cell lineage affects activation states in response to cytokines remains poorly understood. The cytokine environment and cellular infiltrate during an inflammatory response may contain prognostic features that could predict disease outcome. Here we analyzed the transcriptional responses of human monocytes, macrophages, dendritic cells and neutrophils in response to stimulation by IFN-γ, IFN-β IFN-λ, IL-4, IL-13 and IL-10 cytokines, to better understand the heterogeneity of activation states in inflammatory conditions. This generated a myeloid cell cytokine specific response matrix that can infer representation of myeloid cells and the cytokine environment they encounter during infection and in tumors. Neutrophils were highly responsive to type 1 and type 2 cytokine stimulation but did not respond to IL-10. We identified transcripts specific to IFN-β stimulation, whereas other IFN signature genes were upregulated by both IFN-γ and IFN-β. When we used our matrix to deconvolute blood profiles from tuberculosis patients, the IFN-β specific neutrophil signature was reduced in TB patients with active disease whereas the shared response to IFN-γ and IFN-β in neutrophils was increased. When applied to glioma patients, transcripts of neutrophils exposed to IL-4 or IL-13 and monocyte responses to IFN-γ or IFN-β emerged as opposing predictors of patient survival. Hence, by dissecting how different myeloid cells respond to cytokine activation, we can delineate biological roles for myeloid cells in different cytokine environments during disease processes, especially during infection and tumor progression.

Summary Free-living mammals, such as humans and wild mice, display heightened immune activation compared with artificially maintained laboratory mice. These differences are partially attributed to microbial exposure as laboratory mice infected with pathogens exhibit immune profiles more closely resembling that of free-living animals. Here, we examine how colonization by microorganisms within the natural environment contributes to immune system maturation by releasing inbred laboratory mice into an outdoor enclosure. In addition to enhancing differentiation of T cell populations previously associated with pathogen exposure, outdoor release increased circulating granulocytes. However, these “rewilded” mice were not infected by pathogens previously implicated in immune activation. Rather, immune system changes were associated with altered microbiota composition with notable increases in intestinal fungi. Fungi isolated from rewilded mice were sufficient in increasing circulating granulocytes. These findings establish a model to investigate how the natural environment impacts immune development and show that sustained fungal exposure impacts granulocyte numbers.

SUMMARYThe relative contributions of genetic and environmental factors to variation in immune responses are still poorly understood. Here, we performed a deep phenotypic analysis of immunological parameters of laboratory mice released into an outdoor enclosure, carrying susceptibility genes (Nod2 and Atg16l1) implicated in the development of inflammatory bowel diseases. Variations of immune cell populations were largely driven by environment, whereas cytokine production in response to stimulation was affected more by genetic mutations. Multi-omic models identified transcriptional signatures associated with differences in T cell populations. Subnetworks associated with responses against Clostridium perfringens, Candida albicans and Bacteroides vulgatus were also coupled with rewilding. Hence, exposing laboratory mice carrying different genetic mutations to a natural environment uncovered important contributors to immune variation.One sentence summaryNatural environment exposure in laboratory mice primarily promotes variation in population frequencies of immune cells, whereas cytokine responses to stimulation are affected more by genetic susceptibility to inflammatory bowel disease.

Recent advances in mass spectrometry (MS) have enabled extensive analysis of cancer proteomes. Here, we employed quantitative proteomics to profile protein expression across 24 breast cancer patient-derived xenograft (PDX) models. Integrated proteogenomic analysis shows positive correlation between expression measurements from transcriptomic and proteomic analyses; further, gene expression-based intrinsic subtypes are largely re-capitulated using non-stromal protein markers. Proteogenomic analysis also validates a number of predicted genomic targets in multiple receptor tyrosine kinases. However, several protein/phosphoprotein events such as overexpression of AKT proteins and ARAF, BRAF, HSP90AB1 phosphosites are not readily explainable by genomic analysis, suggesting that druggable translational and/or post-translational regulatory events may be uniquely diagnosed by MS. Drug treatment experiments targeting HER2 and components of the PI3K pathway supported proteogenomic response predictions in seven xenograft models. Our study demonstrates that MS-based proteomics can identify therapeutic targets and highlights the potential of PDX drug response evaluation to annotate MS-based pathway activities., National Cancer Institute (U.S.) (Grant R01CA180006)

Inhibition of VLDL secretion reduces plasma levels of atherogenic apolipoprotein B (apoB) lipoproteins but can also cause hepatic steatosis. Approaches targeting apoB synthesis, which lies upstream of VLDL secretion, have potential to effectively reduce dyslipidemia but can also lead to hepatic accumulation of unsecreted triglycerides (TG). Here, we found that treating mice with apoB antisense oligonucleotides (ASOs) for 6 weeks decreased VLDL secretion and plasma cholesterol without causing steatosis. The absence of steatosis was linked to an increase in ER stress in the first 3 weeks of ASO treatment, followed by development of ER autophagy at the end of 6 weeks of treatment. The latter resulted in increased fatty acid (FA) oxidation that was inhibited by both chloroquine and 3-methyl adenine, consistent with trafficking of ER TG through the autophagic pathway before oxidation. These findings support the concept that inhibition of apoB synthesis traps lipids that have been transferred to the ER by microsomal TG transfer protein (MTP), inducing ER stress. ER stress then triggers ER autophagy and subsequent lysosomal lipolysis of TG, followed by mitochondrial oxidation of released FA, leading to prevention of steatosis. The identification of this pathway indicates that inhibition of VLDL secretion remains a viable target for therapies aiming to reduce circulating levels of atherogenic apoB lipoproteins.

Somatic mutations have been extensively characterized in breast cancer, but the effects of these genetic alterations on the proteomic landscape remain poorly understood. Here we describe quantitative mass-spectrometry-based proteomic and phosphoproteomic analyses of 105 genomically annotated breast cancers, of which 77 provided high-quality data. Integrated analyses provided insights into the somatic cancer genome including the consequences of chromosomal loss, such as the 5q deletion characteristic of basal-like breast cancer. Interrogation of the 5q trans-effects against the Library of Integrated Network-based Cellular Signatures, connected loss of CETN3 and SKP1 to elevated expression of epidermal growth factor receptor (EGFR), and SKP1 loss also to increased SRC tyrosine kinase. Global proteomic data confirmed a stromal-enriched group of proteins in addition to basal and luminal clusters, and pathway analysis of the phosphoproteome identified a G-protein-coupled receptor cluster that was not readily identified at the mRNA level. In addition to ERBB2, other amplicon-associated highly phosphorylated kinases were identified, including CDK12, PAK1, PTK2, RIPK2 and TLK2. We demonstrate that proteogenomic analysis of breast cancer elucidates the functional consequences of somatic mutations, narrows candidate nominations for driver genes within large deletions and amplified regions, and identifies therapeutic targets.

We report a comprehensive proteogenomics analysis, including whole-genome sequencing, RNA sequencing, and proteomics and phosphoproteomics profiling, of 218 tumors across 7 histological types of childhood brain cancer: low-grade glioma (n = 93), ependymoma (32), high-grade glioma (25), medulloblastoma (22), ganglioglioma (18), craniopharyngioma (16), and atypical teratoid rhabdoid tumor (12). Proteomics data identify common biological themes that span histological boundaries, suggesting that treatments used for one histological type may be applied effectively to other tumors sharing similar proteomics features. Immune landscape characterization reveals diverse tumor microenvironments across and within diagnoses. Proteomics data further reveal functional effects of somatic mutations and copy number variations (CNVs) not evident in transcriptomics data. Kinase-substrate association and co-expression network analysis identify important biological mechanisms of tumorigenesis. This is the first large-scale proteogenomics analysis across traditional histological boundaries to uncover foundational pediatric brain tumor biology and inform rational treatment selection.

Knowledge of fundamental differences between breast cancer subtypes has driven therapeutic advances; however, basal-like breast cancer (BLBC) remains clinically intractable. Because BLBC exhibits alterations in DNA repair enzymes and cell-cycle checkpoints, elucidation of factors enabling the genomic instability present in this subtype has the potential to reveal novel anti-cancer strategies. Here, we demonstrate that BLBC is especially sensitive to suppression of iron-sulfur cluster (ISC) biosynthesis and identify DNA polymerase epsilon (POLE) as an ISC-containing protein that underlies this phenotype. In BLBC cells, POLE suppression leads to replication fork stalling, DNA damage, and a senescence-like state or cell death. In contrast, luminal breast cancer and non-transformed mammary cells maintain viability upon POLE suppression but become dependent upon an ATR/CHK1/CDC25A/CDK2 DNA damage response axis. We find that CDK1/2 targets exhibit hyperphosphorylation selectively in BLBC tumors, indicating that CDK2 hyperactivity is a genome integrity vulnerability exploitable by targeting POLE.

SUMMARY The integration of mass spectrometry-based proteomics with next-generation DNA and RNA sequencing profiles tumors more comprehensively. Here this “proteogenomics” approach was applied to 122 treatment-naive primary breast cancers accrued to preserve post-translational modifications, including protein phosphorylation and acetylation. Proteogenomics challenged standard breast cancer diagnoses, provided detailed analysis of the ERBB2 amplicon, defined tumor subsets that could benefit from immune checkpoint therapy, and allowed more accurate assessment of Rb status for prediction of CDK4/6 inhibitor responsiveness. Phosphoproteomics profiles uncovered novel associations between tumor suppressor loss and targetable kinases.Acetylproteome analysis highlighted acetylation on key nuclear proteins involved in the DNA damage response and revealed cross-talk between cytoplasmic and mitochondrial acetylation and metabolism. Our results underscore the potential of proteogenomics for clinical investigation of breast cancer through more accurate annotation of targetable pathways and biological features of this remarkably heterogeneous malignancy., In Brief Breast cancer is a highly heterogeneous disease with variable outcomes and subtype-driven treatment approaches, making precision medicine a considerable challenge. Proteogenomic analyses of 122 primary breast cancers provide insights into clinically relevant biology, including cell cycle dysregulation, tumor immunogenicity, aberrant metabolism, and heterogeneity in therapeutic target expression., Graphical Abstract

To explore the biology of lung adenocarcinoma (LUAD) and identify new therapeutic opportunities, we performed comprehensive proteogenomic characterization of 110 tumors and 101 matched normal adjacent tissues (NATs) incorporating genomics, epigenomics, deep-scale proteomics, phosphoproteomics, and acetylproteomics. Multi-omics clustering revealed four subgroups defined by key driver mutations, country, and gender. Proteomic and phosphoproteomic data illuminated biology downstream of copy number aberrations, somatic mutations, and fusions and identified therapeutic vulnerabilities associated with driver events involving KRAS, EGFR, and ALK. Immune subtyping revealed a complex landscape, reinforced the association of STK11 with immune-cold behavior, and underscored a potential immunosuppressive role of neutrophil degranulation. Smoking-associated LUADs showed correlation with other environmental exposure signatures and a field effect in NATs. Matched NATs allowed identification of differentially expressed proteins with potential diagnostic and therapeutic utility. This proteogenomics dataset represents a unique public resource for researchers and clinicians seeking to better understand and treat lung adenocarcinomas.

Background. Multilevel interventions combine individual component interventions, and their design can be informed by decision analysis. Our objective was to identify the optimal combination of interventions for alcohol-using HIV+ individuals on antiretroviral drug therapy in Maharashtra, India, explicitly considering stakeholder constraints. Methods. Using an HIV simulation, we evaluated the expected net monetary benefit (ENMB), the probability of lying on the efficiency frontier (PEF), and annual program costs of 5,836 unique combinations of 15 single-focused HIV risk-reduction interventions. We evaluated scenarios of 1) no constraints (i.e., maximize expected value), 2) short-term budget constraints (limits on annual programmatic costs of US$200,000 and $400,000), and 3) a constraint stemming from risk aversion (requiring that the strategy has >50% PEF). Results. With no constraints, the combination including long individual alcohol counseling, text-message adherence support, long group counseling for sex-risk, and long individual counseling for sex-risk (annual cost = $428,886; PEF ∼27%) maximized ENMB and would be the optimal design. With a cost constraint of $400,000, the combination including long individual alcohol counseling, text-message adherence support, brief group counseling for sex-risk, and long individual counseling for sex-risk (annual cost = $374,745; PEF ∼4%) maximized ENMB. With a cost constraint of $200,000, the combination including long individual alcohol counseling, text-message adherence support, and brief group counseling for sex-risk (annual cost = $187,335; PEF ∼54%) maximized ENMB. With the risk aversion constraint, the same configuration ( long individual alcohol counseling, text-message support, and brief group counseling for sex-risk) maximized health benefit. Conclusion. Evaluating the costs, risks, and projected benefits of alternatives supports informed decision making prior to initiating study; however, stakeholder constraints should be explicitly included and discussed when using decision analyses to guide study design.

Despite the limitations of a small study, our results evidence higher resilience of the gut microbiota with respect to dietary manipulation in adults than in children and urge further studies to understand the extent of microbiota plasticity in response to dietary changes and the mechanisms underlying microbiota resilience. These studies are relevant to the potential of future human pre- and probiotics in preventing or curing microbiota-associated diseases., People living traditional lifestyles have higher gut microbiota diversity than urban subjects. We hypothesized that shifting lifestyles from an urban environment to a traditional rainforest village would lead to changes in the microbiota of visitors, which would become more similar to the microbiota of villagers. Here, we characterized at different time points the microbiota of 7 urban visitors (5 adults and 2 children) staying in a rainforest Amerindian village for 16 days and compared them with a reference collection of samples from age-matched local villagers. We performed a 16S rRNA gene survey of samples from multiple body sites (including fecal, oral, nasal, and skin samples) using Illumina MiSeq sequencing. The main factor segregating the microbiotas of each body site was the human group (i.e., visitors versus villagers), with the visitor microbiota tending to have lower alpha diversity; the lowered alpha diversity was statistically significant in the microbiota of skin and in the children’s fecal and oral microbiota. During the rainforest period, all visitors experienced microbiota changes within their personal cloud of variation. For all body sites, the microbiota conformations in the visitor children better matched the microbiota conformations in villagers of the same age than did those of the visitor adults, which showed a lower “microbiota age” than the microbiota of the villagers. The results suggest higher stability in the adult microbiota, with the less resilient children’s microbiota responding more to dietary changes. IMPORTANCE Despite the limitations of a small study, our results evidence higher resilience of the gut microbiota with respect to dietary manipulation in adults than in children and urge further studies to understand the extent of microbiota plasticity in response to dietary changes and the mechanisms underlying microbiota resilience. These studies are relevant to the potential of future human pre- and probiotics in preventing or curing microbiota-associated diseases.

Alterations in gut microbiota are known to affect intestinal inflammation and obesity. Antibiotic treatment can affect weight gain by elimination of histone deacetylase (HDAC) inhibitor–producing microbes, which are anti-inflammatory by augmenting regulatory T (T(reg)) cells. We asked whether mice that lack HDAC6 and have potent suppressive T(reg) cells are protected from microbiota-induced accelerated weight gain. We crossed wild-type and HDAC6-deficient mice and subjected the offspring to perinatal penicillin, inducing weight gain via microbiota disturbance. We observed that male HDAC6-deficient mice were not protected and developed profoundly accelerated weight gain. The antibiotic-exposed HDAC6-deficient mice showed a mixed immune phenotype with increased CD4(+) and CD8(+) T-cell activation yet maintained the enhanced T(reg) cell–suppressive function phenotype characteristic of HDAC6-deficient mice. 16S rRNA sequencing of mouse fecal samples reveals that their microbiota diverged with time, with HDAC6 deletion altering microbiome composition. On a high-fat diet, HDAC6-deficient mice were depleted in representatives of the S24-7 family and Lactobacillus but enriched with Bacteroides and Parabacteroides; these changes are associated with obesity. Our findings further our understanding of the influence of HDACs on microbiome composition and are important for the development of HDAC6 inhibitors in the treatment of human diseases.—Lieber, A. D., Beier, U. H., Xiao, H., Wilkins, B. J., Jiao, J., Li, X. S., Schugar, R. C., Strauch, C. M., Wang, Z., Brown, J. M., Hazen, S. L., Bokulich, N. A., Ruggles, K. V., Akimova, T., Hancock, W. W., Blaser, M. J. Loss of HDAC6 alters gut microbiota and worsens obesity.

Translation initiation of most mammalian mRNAs is mediated by a 5′ cap structure that binds eukaryotic initiation factor 4E (eIF4E). However, inactivation of eIF4E does not impair translation of many capped mRNAs, suggesting an unknown alternate mechanism may exist for cap-dependent but eIF4E-independent translation. We show that DAP5, an eIF4GI homolog that lacks eIF4E binding, utilizes eIF3d to facilitate cap-dependent translation of approximately 20% of mRNAs. Genome-wide transcriptomic and translatomic analyses indicate that DAP5 is required for translation of many transcription factors and receptor capped mRNAs and their mRNA targets involved in cell survival, motility, DNA repair and translation initiation, among other mRNAs. Mass spectrometry and crosslinking studies demonstrate that eIF3d is a direct binding partner of DAP5. In vitro translation and ribosome complex studies demonstrate that DAP5 and eIF3d are both essential for eIF4E-independent capped-mRNA translation. These studies disclose a widespread and previously unknown mechanism for cap-dependent mRNA translation by DAP5-eIF3d complexes., Binding of eIF4E to the 5′ cap of mRNAs is a key early step in canonical translation initiation, but the requirement for eIF4E is not universal. Here the authors show that the eIF4G homolog DAP5 interacts with eIF3 to promote cap-dependent translation of a significant number of mRNA in an eIF4E-independent manner.

The early-life intestinal microbiota plays a key role in shaping host immune system development. We found that a single early-life antibiotic course (1PAT) accelerated type 1 diabetes (T1D) development in male NOD mice. The single course had deep and persistent effects on the intestinal microbiome, leading to altered cecal, hepatic, and serum metabolites. The exposure elicited sex-specific effects on chromatin states in the ileum and liver and perturbed ileal gene expression, altering normal maturational patterns. The global signature changes included specific genes controlling both innate and adaptive immunity. Microbiome analysis revealed four taxa each that potentially protect against or accelerate T1D onset, that were linked in a network model to specific differences in ileal gene expression. This simplified animal model reveals multiple potential pathways to understand pathogenesis by which early-life gut microbiome perturbations alter a global suite of intestinal responses, contributing to the accelerated and enhanced T1D development., eLife digest The human body contains many microbes that play important roles in our health. These microbes begin to live in the intestines, skin, and mouth shortly after birth. They form complex communities called the microbiome, which changes as babies develop. The microbiome works with organs to maintain human health. For example, the lower intestinal tract is home to the most numerous and active microbes in the body. The intestines provide microbes with food and a welcoming environment, and the microbes make products the body needs, influence immune system development, and help maintain a balance of beneficial microbes. Use of antibiotics to treat infections, particularly early in life, disrupts intestinal microbe communities. Recent studies show that such microbiome disturbances may affect how the immune system develops and the rate at which type 1 diabetes develops. Type 1 diabetes is an autoimmune disease in which the immune system destroys cells in the pancreas that produce insulin. Scientists would like to learn more about how use of antibiotics in early life may contribute to the development of this disease. Now, Zhang et al. show that a single course of antibiotics administered early in life accelerates the development of type 1 diabetes in mice prone to develop the disease. In the experiments, a strain of laboratory mice that spontaneously develops type 1 diabetes were either given a single course of antibiotics, three courses of antibiotics, or no antibiotics in their first weeks of life. After one single course, the gut microbiome was different in mice treated with antibiotics compared with mice who were never exposed. The antibiotics also changed the molecules produced by these microbes. These alterations in the microbiome turned on or off certain genes in the intestine, affecting the development of the immune system. Zhang et al. identified some microbes that appear to protect against type 1 diabetes and others that seem to speed it up and how they do so. Antibiotic use in children is very common, so finding ways to reduce its potentially harmful effects on development are critical. The experiments provide one way to study how antibiotics may contribute to autoimmune disease. It also may allow scientists to test ways to reverse harmful change.

Background Translatomics data, particularly genome-wide ribosome profiling and polysome profiling, provide multiple levels of gene regulatory information that can be used to assess general transcription and translation, as well translational efficiency. The increasing popularity of these techniques has resulted in multiple algorithms to detect translational regulation, typically distributed in the form of command line tools that require a basic level of programming ability. Additionally, due to the static nature of current software, dynamic transcriptional and translational comparative analysis cannot be adequately achieved. In order to streamline hypothesis generation, investigators must have the ability to manipulate and interact with their data in real-time. Results To address the lack of integration in current software, we introduce RIVET, Ribosomal Investigation and Visualization to Evaluate Translation, an R shiny based graphical user interface for translatomics data exploration and differential analysis. RIVET can analyze either microarray or RNA sequencing data from polysome profiling and ribosome profiling experiments. RIVET provides multiple choices for statistical analysis as well as integration of transcription, translation, and translational efficiency data analytics and the ability to visualize all results dynamically. Conclusions RIVET is a user-friendly tool designed for bench scientists with little to no programming background. RIVET facilitates the data analysis of translatomics data allowing for dynamic generation of results based on user-defined inputs and publication ready visualization. We expect RIVET will allow for scientists to efficiently make more comprehensive data observations that will lead to more robust hypothesis regarding translational regulation. Electronic supplementary material The online version of this article (10.1186/s12864-018-5166-z) contains supplementary material, which is available to authorized users.

Background Cancers induce gene expression alterations in stroma surrounding tumors that supports cancer progression. However, it is actually not at all known the extent of altered stromal gene expression enacted by tumors nor the extent to which altered stromal gene expression penetrates the stromal tissue. Presently, post-surgical “tumor-free” stromal tissue is determined to be cancer-free based on solely on morphological normality—a criteria that has not changed in more than 100 years despite the existence of sophisticated gene expression data to the contrary. We therefore investigated the extent to which breast tumors alter stromal gene expression in three dimensions in women undergoing mastectomy with the intent of providing a genomic determination for development of future risk of recurrence criteria, and to inform the need for adjuvant full-breast irradiation. Methods and findings Genome-wide gene expression changes were determined in histopathologically normal breast tissue in 33 women undergoing mastectomy for stage II and III primary invasive ductal carcinoma at serial distances in three dimensions from the tumor. Gene expression was determined by genome-wide mRNA analysis and subjected to metagene mRNA characterization. Tumor-like gene expression signatures in stroma were identified that surprisingly transitioned to a plastic, normalizing homeostatic signature with distance from tumor. Stroma closest to tumor displayed a pronounced tumor-like signature enriched in cancer-promoting pathways involved in disruption of basement membrane, cell migration and invasion, WNT signaling and angiogenesis. By 2 cm from tumor in all dimensions, stromal tissues were in transition, displaying homeostatic and tumor suppressing gene activity, while also expressing cancer supporting pathways. Conclusions The dynamics of gene expression in the post-tumor breast stroma likely co-determines disease outcome: reversion to normality or transition to transformation in morphologically normal tissue. Our stromal genomic signature may be important for personalizing surgical and adjuvant therapeutic decisions and risk of recurrence.

Background Exploration of large data sets, such as shotgun metagenomic sequence or expression data, by biomedical experts and medical professionals remains as a major bottleneck in the scientific discovery process. Although tools for this purpose exist for 16S ribosomal RNA sequencing analysis, there is a growing but still insufficient number of user-friendly interactive visualization workflows for easy data exploration and figure generation. The development of such platforms for this purpose is necessary to accelerate and streamline microbiome laboratory research. Results We developed the Workflow Hub for Automated Metagenomic Exploration (WHAM!) as a web-based interactive tool capable of user-directed data visualization and statistical analysis of annotated shotgun metagenomic and metatranscriptomic data sets. WHAM! includes exploratory and hypothesis-based gene and taxa search modules for visualizing differences in microbial taxa and gene family expression across experimental groups, and for creating publication quality figures without the need for command line interface or in-house bioinformatics. Conclusions WHAM! is an interactive and customizable tool for downstream metagenomic and metatranscriptomic analysis providing a user-friendly interface allowing for easy data exploration by microbiome and ecological experts to facilitate discovery in multi-dimensional and large-scale data sets. Electronic supplementary material The online version of this article (10.1186/s12864-018-4870-z) contains supplementary material, which is available to authorized users.

Improvements in mass spectrometry (MS)-based peptide sequencing provide a new opportunity to determine whether polymorphisms, mutations, and splice variants identified in cancer cells are translated. Herein, we apply a proteogenomic data integration tool (QUILTS) to illustrate protein variant discovery using whole genome, whole transcriptome, and global proteome datasets generated from a pair of luminal and basal-like breast-cancer-patient-derived xenografts (PDX). The sensitivity of proteogenomic analysis for singe nucleotide variant (SNV) expression and novel splice junction (NSJ) detection was probed using multiple MS/MS sample process replicates defined here as an independent tandem MS experiment using identical sample material. Despite analysis of over 30 sample process replicates, only about 10% of SNVs (somatic and germline) detected by both DNA and RNA sequencing were observed as peptides. An even smaller proportion of peptides corresponding to NSJ observed by RNA sequencing were detected (

Every molecular player in the cast of biology’s central dogma is being sequenced and quantified with increasing ease and coverage. To bring the resulting genomic, transcriptomic, and proteomic data sets into coherence, tools must be developed that do not constrain data acquisition and analytics in any way but rather provide simple links across previously acquired data sets with minimal preprocessing and hassle. Here we present such a tool: PGx, which supports proteogenomic integration of mass spectrometry proteomics data with next-generation sequencing by mapping identified peptides onto their putative genomic coordinates.

Bottom-up proteomics relies on the use of proteases and is the method of choice for identifying thousands of protein groups in complex samples. Top-down proteomics has been shown to be robust for direct analysis of small proteins and offers a solution to the "peptide-to-protein" inference problem inherent with bottom-up approaches. Here, we describe the first large-scale integration of genomic, bottom-up and top-down proteomic data for the comparative analysis of patient-derived mouse xenograft models of basal and luminal B human breast cancer, WHIM2 and WHIM16, respectively. Using these well-characterized xenograft models established by the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium, we compared and contrasted the performance of bottom-up and top-down proteomics to detect cancer-specific aberrations at the peptide and proteoform levels and to measure differential expression of proteins and proteoforms. Bottom-up proteomic analysis of the tumor xenografts detected almost 10 times as many coding nucleotide polymorphisms and peptides resulting from novel splice junctions than top-down. For proteins in the range of 0-30 kDa, where quantitation was performed using both approaches, bottom-up proteomics quantified 3,519 protein groups from 49,185 peptides, while top-down proteomics quantified 982 proteoforms mapping to 358 proteins. Examples of both concordant and discordant quantitation were found in a ∼60:40 ratio, providing a unique opportunity for top-down to fill in missing information. The two techniques showed complementary performance, with bottom-up yielding eight times more identifications of 0-30 kDa proteins in xenograft proteomes, but failing to detect differences in certain posttranslational modifications (PTMs), such as phosphorylation pattern changes of alpha-endosulfine. This work illustrates the potency of a combined bottom-up and top-down proteomics approach to deepen our knowledge of cancer biology, especially when genomic data are available.

Drug and alcohol use have been associated with increased risk for sexual violence, but there is little research on sexual violence within the context of drug use among young adult opioid users. The current mixed-methods study explores young adult opioid users’ sexual experiences in the context of their drug use. Forty-six New York City young adults (ages 18-32) who reported lifetime nonmedical use of prescription opioids (POs) completed in-depth, semistructured interviews, and 164 (ages 18-29) who reported heroin and/or nonmedical PO use in the past 30 days completed structured assessments that inquired about their drug use and sexual behavior and included questions specific to sexual violence. Participants reported frequent incidents of sexual violence experienced both personally and by their opioid using peers. Participants described sexual violence, including sexual assault, as occurring within a context characterized by victimization of users who were unconscious as a result of substance use, implicit and explicit exchanges of sex for drugs and/or money that increased risk for sexual violence, negative sexual perceptions ascribed to drug users, and participants’ own internalized stigma. Recommendations to reduce sexual violence among young adult opioid users include education for users and service providers on the risk of involvement in sexual violence within drug using contexts and efforts to challenge perceptions of acceptability regarding sexual violence.

A key component of eukaryotic lipid homeostasis is the esterification of sterols with fatty acids by sterol O-acyltransferases (SOATs). The esterification reactions are allosterically activated by their sterol substrates, the majority of which accumulate at the plasma membrane. We demonstrate that in yeast, sterol transport from the plasma membrane to the site of esterification is associated with the physical interaction of the major SOAT, acyl-coenzyme A:cholesterol acyltransferase (ACAT)-related enzyme (Are)2p, with 2 plasma membrane ATP-binding cassette (ABC) transporters: Aus1p and Pdr11p. Are2p, Aus1p, and Pdr11p, unlike the minor acyltransferase, Are1p, colocalize to sterol and sphingolipid-enriched, detergent-resistant microdomains (DRMs). Deletion of either ABC transporter results in Are2p relocalization to detergent-soluble membrane domains and a significant decrease (53–36%) in esterification of exogenous sterol. Similarly, in murine tissues, the SOAT1/Acat1 enzyme and activity localize to DRMs. This subcellular localization is diminished upon deletion of murine ABC transporters, such as Abcg1, which itself is DRM associated. We propose that the close proximity of sterol esterification and transport proteins to each other combined with their residence in lipid-enriched membrane microdomains facilitates rapid, high-capacity sterol transport and esterification, obviating any requirement for soluble intermediary proteins.—Gulati, S., Balderes, D., Kim, C., Guo, Z. A., Wilcox, L., Area-Gomez, E., Snider, J., Wolinski, H., Stagljar, I., Granato, J. T., Ruggles, K. V., DeGiorgis, J. A., Kohlwein, S. D., Schon, E. A., Sturley, S. L. ATP-binding cassette transporters and sterol O-acyltransferases interact at membrane microdomains to modulate sterol uptake and esterification.

Activation of PI3K signaling is frequently observed in triple-negative breast cancer (TNBC), yet PI3K inhibitors have shown limited clinical activity. To investigate intrinsic and adaptive mechanisms of resistance, we analyzed a panel of patient-derived xenograft models of TNBC with varying responsiveness to buparlisib, a pan-PI3K inhibitor. In a subset of patient-derived xenografts, resistance was associated with incomplete inhibition of PI3K signaling and upregulated MAPK/MEK signaling in response to buparlisib. Outlier phosphoproteome and kinome analyses identified novel candidates functionally important to buparlisib resistance, including NEK9 and MAP2K4. Knockdown of NEK9 or MAP2K4 reduced both baseline and feedback MAPK/MEK signaling and showed synthetic lethality with buparlisib in vitro. A complex in/del frameshift in PIK3CA decreased sensitivity to buparlisib via NEK9/MAP2K4–dependent mechanisms. In summary, our study supports a role for NEK9 and MAP2K4 in mediating buparlisib resistance and demonstrates the value of unbiased omic analyses in uncovering resistance mechanisms to targeted therapy. Significance: Integrative phosphoproteogenomic analysis is used to determine intrinsic resistance mechanisms of triple-negative breast tumors to PI3K inhibition. Cancer Res; 78(10); 2732–46. ©2018 AACR.

OBJECTIVES We identified the prevalence of nonmedical prescription drug use and its relationship to heroin and injection drug use in 4 nationally representative samples of adolescents. METHODS We used the most recent data (2009-2015) from the Youth Risk Behavior Surveillance System (Ntotal= 61,132). Prevalence rates and 95% confidence intervals for prescription drug misuse, heroin use, and injection drug use were calculated across time points, sex, and race/ethnicity subgroups. Using odds ratios, we determined the likelihood of youth reporting nonmedical prescription drug use also reporting heroin and drug injection. RESULTS In 2015, one in 6 adolescents reported recent prescription drug misuse. High rates of nonmedical prescription drug use persisted or increased among Hispanic boys, black boys, and "other" youth, while declining among white youth. Youth who used prescription drugs nonmedically at least once were 17.5 times more likely to have used heroin (CI: 13.7, 22.4) and 14.6 times more likely to have injected drugs (CI: 11.2, 19.2) in their lifetime. CONCLUSIONS Public health programming focused on reducing prescription drug misuse also may reduce youth engagement in heroin and/or injection drug use. Preventive efforts to support communities of color in reducing rates of prescription drug misuse are crucial.

Increased intestinal permeability and translocation of gut bacteria trigger various polyaetiological diseases associated with chronic inflammation and underlie a variety of poorly treatable pathologies. Previous studies have established a primary role of the microbiota composition and intestinal permeability in such pathologies. Using a rat model, we examined the effects of exposure to a bacteriophage cocktail on intestinal permeability and relative abundance of taxonomic units in the gut bacterial community. There was an increase in markers of impaired gut permeability, such as the lactulose/mannitol ratio, plasma endotoxin concentrations, and serum levels of inflammation-related cytokines, following the bacteriophage challenge. We observed significant differences in the alpha diversity of faecal bacterial species and found that richness and diversity index values increased following the bacteriophage challenge. There was a reduction in the abundance of Blautia, Catenibacterium, Lactobacillus, and Faecalibacterium species and an increase in Butyrivibrio, Oscillospira and Ruminococcus after bacteriophage administration. These findings provide novel insights into the role of bacteriophages as potentially pathogenic for mammals and their possible implication in the development of diseases associated with increased intestinal permeability.

Nonmedical prescription opioid use has become widespread. It can lead to heroin use, drug injection and HIV infection. We describe young adult opioid users’ sexual risk behavior, partnerships and settings. 464 youth aged 18–29 who reported opioid use in the past 30 days were recruited using Respondent-Driven Sampling. Eligible participants completed a computer-assisted, interviewer-administered risk questionnaire and were tested for STIs and HIV. Participants (33% female; 66% white non-Hispanic) almost all had sex in the prior 90 days; 42% reported more than one partner. Same-sex sex was reported by 3% of men and 10% of women. Consistent condom use was rare. Seven percent reported group sex participation in the last 90 days but lifetime group sex was common among men and women. Young opioid users’ unprotected sex, multiple partners and group sex puts them and others at high HIV and STI risk.

We undertook a comprehensive proteogenomic characterization of 95 prospectively collected endometrial carcinomas, comprising 83 endometrioid and 12 serous tumors. This analysis revealed possible new consequences of perturbations to the p53 and Wnt/β-catenin pathways, identified a potential role for circRNAs in the epithelial-mesenchymal transition, and provided new information about proteomic markers of clinical and genomic tumor subgroups, including relationships to known druggable pathways. An extensive genome-wide acetylation survey yielded insights into regulatory mechanisms linking Wnt signaling and histone acetylation. We also characterized aspects of the tumor immune landscape, including immunogenic alterations, neoantigens, common cancer/testis antigens, and the immune microenvironment, all of which can inform immunotherapy decisions. Collectively, our multi-omic analyses provide a valuable resource for researchers and clinicians, identify new molecular associations of potential mechanistic significance in the development of endometrial cancers, and suggest novel approaches for identifying potential therapeutic targets.

SUMMARY To elucidate the deregulated functional modules that drive clear cell renal cell carcinoma (ccRCC), we performed comprehensive genomic, epigenomic, transcriptomic, proteomic, and phosphoproteomic characterization of treatment-naive ccRCC and paired normal adjacent tissue samples. Genomic analyses identified a distinct molecular subgroup associated with genomic instability. Integration of proteogenomic measurements uniquely identified protein dysregulation of cellular mechanisms impacted by genomic alterations, including oxidative phosphorylation-related metabolism, protein translation processes, and phospho-signaling modules. To assess the degree of immune infiltration in individual tumors, we identified microenvironment cell signatures that delineated four immune-based ccRCC subtypes characterized by distinct cellular pathways. This study reports a large-scale proteogenomic analysis of ccRCC to discern the functional impact of genomic alterations and provides evidence for rational treatment selection stemming from ccRCC pathobiology., Graphical Abstract, In Brief Comprehensive proteogenomic characterization in 103 treatment-naive clear cell renal cell carcinoma patient samples highlights tumor-specific alterations at the proteomic level that are unrevealed by transcriptomic profiling and proposes a revised subtyping scheme based on integrated omics analysis.

We performed the first proteogenomic study on a prospectively collected colon cancer cohort. Comparative proteomic and phosphoproteomic analysis of paired tumor and normal adjacent tissues produced a catalog of colon cancer-associated proteins and phosphosites, including known and putative new biomarkers, drug targets, and cancer/testis antigens. Proteogenomic integration not only prioritized genomically inferred targets, such as copy-number drivers and mutation-derived neoantigens, but also yielded novel findings. Phosphoproteomics data associated Rb phosphorylation with increased proliferation and decreased apoptosis in colon cancer, which explains why this classical tumor suppressor is amplified in colon tumors and suggests a rationale for targeting Rb phosphorylation in colon cancer. Proteomics identified an association between decreased CD8 T cell infiltration and increased glycolysis in microsatellite instability-high (MSI-H) tumors, suggesting glycolysis as a potential target to overcome the resistance of MSI-H tumors to immune checkpoint blockade. Proteogenomics presents new avenues for biological discoveries and therapeutic development.