One-pot method for preparing DNA, RNA, and protein for multiomics analysis.

Typical multiomics studies employ separate methods for DNA, RNA, and protein sample preparation, which is labor intensive, costly, and prone to sampling bias. We describe a method for preparing high-quality, sequencing-ready DNA and RNA, and either intact proteins or mass-spectrometry-ready peptides for whole proteome analysis from a single sample. This method utilizes a reversible protein tagging scheme to covalently link all proteins in a lysate to a bead-based matrix and nucleic acid precipitation and selective solubilization to yield separate pools of protein and nucleic acids. We demonstrate the utility of this method to compare the genomes, transcriptomes, and proteomes of four triple-negative breast cancer cell lines with different degrees of malignancy. These data show the involvement of both RNA and associated proteins, and protein-only dependent pathways that distinguish these cell lines. We also demonstrate the utility of this multiomics workflow for tissue analysis using mouse brain, liver, and lung tissue.

Exosomal MicroRNA and Protein Profiles of Hepatitis B Virus-Related Hepatocellular Carcinoma Cells.

Infection with hepatitis B virus (HBV) is a main risk factor for hepatocellular carcinoma (HCC). Extracellular vesicles, such as exosomes, play an important role in tumor development and metastasis, including regulation of HBV-related HCC. In this study, we have characterized exosome microRNA and proteins released in vitro from hepatitis B virus (HBV)-related HCC cell lines SNU-423 and SNU-182 and immortalized normal hepatocyte cell lines (THLE2 and THLE3) using microRNA sequencing and mass spectrometry. Bioinformatics, including functional enrichment and network analysis, combined with survival analysis using data related to HCC in The Cancer Genome Atlas (TCGA) database, were applied to examine the prognostic significance of the results. More than 40 microRNAs and 200 proteins were significantly dysregulated (p < 0.05) in the exosomes released from HCC cells in comparison with the normal liver cells. The functional analysis of the differentially expressed exosomal miRNAs (i.e., mir-483, mir-133a, mir-34a, mir-155, mir-183, mir-182), their predicted targets, and exosomal differentially expressed proteins (i.e., POSTN, STAM, EXOC8, SNX9, COL1A2, IDH1, FN1) showed correlation with pathways associated with HBV, virus activity and invasion, exosome formation and adhesion, and exogenous protein binding. The results from this study may help in our understanding of the role of HBV infection in the development of HCC and in the development of new targets for treatment or non-invasive predictive biomarkers of HCC.

Repurposing live attenuated trivalent MMR vaccine as cost-effective cancer immunotherapy.

It has long been known that oncolytic viruses wield their therapeutic capability by priming an inflammatory state within the tumor and activating the tumor immune microenvironment, resulting in a multifaceted antitumor immune response. Vaccine-derived viruses, such as measles and mumps, have demonstrated promising potential for treating human cancer in animal models and clinical trials. However, the extensive cost of manufacturing current oncolytic viral products makes them far out of reach for most patients. Here by analyzing the impact of intratumoral (IT) administrations of the trivalent live attenuated measles, mumps, and rubella viruses (MMR) vaccine, we unveil the cellular and molecular basis of MMR-induced anti-cancer activity. Strikingly, we found that IT delivery of low doses of MMR correlates with tumor control and improved survival in murine hepatocellular cancer and colorectal cancer models via increased tumor infiltration of CD8+ granzyme B+ T-cells and decreased macrophages. Moreover, our data indicate that MMR activates key cellular effectors of the host's innate and adaptive antitumor immunity, culminating in an immunologically coordinated cancer cell death. These findings warrant further work on the potential for MMR to be repurposed as safe and cost-effective cancer immunotherapy to impact cancer patients globally.

In vivo transcriptional analysis of mice infected with Leishmania major unveils cellular heterogeneity and altered transcriptomic profiling at single-cell resolution.

Leishmania parasites cause cutaneous leishmaniasis (CL), a disease characterized by disfiguring, ulcerative skin lesions. Both parasite and host gene expression following infection with various Leishmania species has been investigated in vitro, but global transcriptional analysis following L. major infection in vivo is lacking. Thus, we conducted a comprehensive transcriptomic profiling study combining bulk RNA sequencing (RNA-Seq) and single-cell RNA sequencing (scRNA-Seq) to identify global changes in gene expression in vivo following L. major infection. Bulk RNA-Seq analysis revealed that host immune response pathways like the antigen processing and presentation pathway were significantly enriched amongst differentially expressed genes (DEGs) upon infection, while ribosomal pathways were significantly downregulated in infected mice compared to naive controls. scRNA-Seq analyses revealed cellular heterogeneity including distinct resident and recruited cell types in the skin following murine L. major infection. Within the individual immune cell types, several DEGs indicative of many interferon induced GTPases and antigen presentation molecules were significantly enhanced in the infected ears including macrophages, resident macrophages, and inflammatory monocytes. Ingenuity Pathway Analysis of scRNA-Seq data indicated the antigen presentation pathway was increased with infection, while EIF2 signaling is the top downregulated pathway followed by eIF4/p70S6k and mTOR signaling in multiple cell types including macrophages, blood and lymphatic endothelial cells. Altogether, this transcriptomic profile highlights known recruitment of myeloid cells to lesions and recognizes a potential role for EIF2 signaling in murine L. major infection in vivo.

Proteomic profiling of tear fluid as a promising non-invasive screening test for colon cancer.

BACKGROUND: Current screening options for colorectal cancer (CRC) are either invasive (colonoscopy) or have lower sensitivity to identify pre-malignant lesions (fecal immunochemical test). We proposed to identify protein profiles in tears of patients with both pre-malignant polyps and CRC; these profiles could have potential as a noninvasive screening test. METHOD: Colonoscopy patients were divided into "high risk" group (CRC and tubular adenomatous polyp) and "low risk" (normal and hyperplastic polyps). Tear fluids from patients were analyzed by Liquid Chromatography Mass Spectrometry/Mass Spectrometry. The data were analyzed for protein expression, protein-protein interaction and gene set enrichment. RESULTS: The results showed 80 proteins (18 up-regulated and 62 down-regulated) significantly differentiated in "high-risk" compared to "low-risk"; Twenty-eight of these show protein-protein interactions, 9 of which were associated with pathways demonstrated to be altered in CRC patients. CONCLUSION: Our pilot data, though limited, demonstrated tear protein profiling could distinguish the groups of patients with and without colon lesions.

HIF-α Activation Impacts Macrophage Function during Murine Leishmania major Infection.

Leishmanial skin lesions are characterized by inflammatory hypoxia alongside the activation of hypoxia-inducible factors, HIF-1α and HIF-2α, and subsequent expression of the HIF-α target VEGF-A during Leishmania major infection. However, the factors responsible for HIF-α activation are not known. We hypothesize that hypoxia and proinflammatory stimuli contribute to HIF-α activation during infection. RNA-Seq of leishmanial lesions revealed that transcripts associated with HIF-1α signaling were induced. To determine whether hypoxia contributes to HIF-α activation, we followed the fate of myeloid cells infiltrating from the blood and into hypoxic lesions. Recruited myeloid cells experienced hypoxia when they entered inflamed lesions, and the length of time in lesions increased their hypoxic signature. To determine whether proinflammatory stimuli in the inflamed tissue can also influence HIF-α activation, we subjected macrophages to various proinflammatory stimuli and measured VEGF-A. While parasites alone did not induce VEGF-A, and proinflammatory stimuli only modestly induced VEGF-A, HIF-α stabilization increased VEGF-A during infection. HIF-α stabilization did not impact parasite entry, growth, or killing. Conversely, the absence of ARNT/HIF-α signaling enhanced parasite internalization. Altogether, these findings suggest that HIF-α is active during infection, and while macrophage HIF-α activation promotes lymphatic remodeling through VEGF-A production, HIF-α activation does not impact parasite internalization or control.

Phosphoproteomics Provides Novel Insights into the Response of Primary Acute Lymphoblastic Leukemia Cells to Microtubule Depolymerization in G1 Phase of the Cell Cycle.

Microtubule targeting agents (MTAs) have been used for the treatment of cancer for many decades and are among the most successful chemotherapeutic agents. However, their application and effectiveness are limited because of toxicity and resistance as well as a lack of knowledge of molecular mechanisms downstream of microtubule inhibition. Insights into key pathways that link microtubule disruption to cell death is critical for optimal use of these drugs, for defining biomarkers useful in patient stratification, and for informed design of drug combinations. Although MTAs characteristically induce death in mitosis, microtubule destabilizing agents such as vincristine also induce death directly in G1 phase in primary acute lymphoblastic leukemia (ALL) cells. Because many signaling pathways regulating cell survival and death involve changes in protein expression and phosphorylation, we undertook a comprehensive quantitative proteomic study of G1 phase ALL cells treated with vincristine. The results revealed distinct alterations associated with c-Jun N-terminal kinase signaling, anti-proliferative signaling, the DNA damage response, and cytoskeletal remodeling. Signals specifically associated with cell death were identified by pre-treatment with the CDK4/6 inhibitor palbociclib, which caused G1 arrest and precluded death induction. These results provide insights into signaling mechanisms regulating cellular responses to microtubule inhibition and provide a foundation for a better understanding of the clinical mechanisms of MTAs and for the design of novel drug combinations. The mass spectrometry proteomics data have been deposited to the PRIDE Archive (http://www.ebi.ac.uk/pride/archive/) via the PRIDE partner repository with the data set identifier PXD027190 and 10.6019/PXD027190.

Multi-omics data integration reveals correlated regulatory features of triple negative breast cancer.

Triple negative breast cancer (TNBC) is an aggressive type of breast cancer with very little treatment options. TNBC is very heterogeneous with large alterations in the genomic, transcriptomic, and proteomic landscapes leading to various subtypes with differing responses to therapeutic treatments. We applied a multi-omics data integration method to evaluate the correlation of important regulatory features in TNBC BRCA1 wild-type MDA-MB-231 and TNBC BRCA1 5382insC mutated HCC1937 cells compared with non-tumorigenic epithelial breast MCF10A cells. The data includes DNA methylation, RNAseq, protein, phosphoproteomics, and histone post-translational modification. Data integration methods identified regulatory features from each omics method that had greater than 80% positive correlation within each TNBC subtype. Key regulatory features at each omics level were identified distinguishing the three cell lines and were involved in important cancer related pathways such as TGFß signaling, PI3K/AKT/mTOR, and Wnt/beta-catenin signaling. We observed overexpression of PTEN, which antagonizes the PI3K/AKT/mTOR pathway, and MYC, which downregulates the same pathway in the HCC1937 cells relative to the MDA-MB-231 cells. The PI3K/AKT/mTOR and Wnt/beta-catenin pathways are both downregulated in HCC1937 cells relative to MDA-MB-231 cells, which likely explains the divergent sensitivities of these cell lines to inhibitors of downstream signaling pathways. The DNA methylation and RNAseq data is freely available via GEO GSE171958 and the proteomics data is available via the ProteomeXchange PXD025238.

PTMViz: a tool for analyzing and visualizing histone post translational modification data.

BACKGROUND: Histone post-translational modifications (PTMs) play an important role in our system by regulating the structure of chromatin and therefore contribute to the regulation of gene and protein expression. Irregularities in histone PTMs can lead to a variety of different diseases including various forms of cancer. Histone modifications are analyzed using high resolution mass spectrometry, which generate large amounts of data that requires sophisticated bioinformatics tools for analysis and visualization. PTMViz is designed for downstream differential abundance analysis and visualization of both protein and/or histone modifications. RESULTS: PTMViz provides users with data tables and visualization plots of significantly differentiated proteins and histone PTMs between two sample groups. All the data is packaged into interactive data tables and graphs using the Shiny platform to help the user explore the results in a fast and efficient manner to assess if changes in the system are due to protein abundance changes or epigenetic changes. In the example data provided, we identified several proteins differentially regulated in the dopaminergic pathway between mice treated with methamphetamine compared to a saline control. We also identified histone post-translational modifications including histone H3K9me, H3K27me3, H4K16ac, and that were regulated due to drug exposure. CONCLUSIONS: Histone modifications play an integral role in the regulation of gene expression. PTMViz provides an interactive platform for analyzing proteins and histone post-translational modifications from mass spectrometry data in order to quickly identify differentially expressed proteins and PTMs.

PHF19 inhibition as a therapeutic target in multiple myeloma.

Epigenetic deregulation is increasingly recognized as a contributing pathological factor in multiple myeloma (MM). In particular tri-methylation of H3 lysine 27 (H3K27me3), which is catalyzed by PHD finger protein 19 (PHF19), a subunit of the Polycomb Repressive Complex 2 (PRC2), has recently shown to be a crucial mediator of MM tumorigenicity. Overexpression of PHF19 in MM has been associated with worse clinical outcome. Yet, while there is mounting evidence that PHF19 overexpression plays a crucial role in MM carcinogenesis downstream mechanisms remain to be elucidated. In the current study we use a functional knock down (KD) of PHF19 to investigate the biological role of PHF19 and show that PHF19KD leads to decreased tumor growth in vitro and in vivo. Expression of major cancer players such as bcl2, myc and EGR1 were decreased upon PHF19KD further underscoring the role of PHF19 in MM biology. Additionally, our results highlighted the prognostic impact of PHF19 overexpression, which was significantly associated with worse survival. Overall, our study underscores the premise that targeting the PHF19-PRC2 complex would open up avenues for novel MM therapies.

Multi-omics data integration considerations and study design for biological systems and disease.

With the advancement of next-generation sequencing and mass spectrometry, there is a growing need for the ability to merge biological features in order to study a system as a whole. Features such as the transcriptome, methylome, proteome, histone post-translational modifications and the microbiome all influence the host response to various diseases and cancers. Each of these platforms have technological limitations due to sample preparation steps, amount of material needed for sequencing, and sequencing depth requirements. These features provide a snapshot of one level of regulation in a system. The obvious next step is to integrate this information and learn how genes, proteins, and/or epigenetic factors influence the phenotype of a disease in context of the system. In recent years, there has been a push for the development of data integration methods. Each method specifically integrates a subset of omics data using approaches such as conceptual integration, statistical integration, model-based integration, networks, and pathway data integration. In this review, we discuss considerations of the study design for each data feature, the limitations in gene and protein abundance and their rate of expression, the current data integration methods, and microbiome influences on gene and protein expression. The considerations discussed in this review should be regarded when developing new algorithms for integrating multi-omics data.

Epigenetic Control of Cdkn2a.Arf Protects Tumor-Infiltrating Lymphocytes from Metabolic Exhaustion.

T-cell exhaustion in cancer is linked to poor clinical outcomes, where evidence suggests T-cell metabolic changes precede functional exhaustion. Direct competition between tumor-infiltrating lymphocytes (TIL) and cancer cells for metabolic resources often renders T cells dysfunctional. Environmental stress produces epigenome remodeling events within TIL resulting from loss of the histone methyltransferase EZH2. Here, we report an epigenetic mechanism contributing to the development of metabolic exhaustion in TIL. A multiomics approach revealed a Cdkn2a.Arf-mediated, p53-independent mechanism by which EZH2 inhibition leads to mitochondrial dysfunction and the resultant exhaustion. Reprogramming T cells to express a gain-of-function EZH2 mutant resulted in an enhanced ability of T cells to inhibit tumor growth in vitro and in vivo. Our data suggest that manipulation of T-cell EZH2 within the context of cellular therapies may yield lymphocytes that are able to withstand harsh tumor metabolic environments and collateral pharmacologic insults. SIGNIFICANCE: These findings demonstrate that manipulation of T-cell EZH2 in cellular therapies may yield cellular products able to withstand solid tumor metabolic-deficient environments. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/21/4707/F1.large.jpg.

Formula Diet Alters the Ileal Metagenome and Transcriptome at Weaning and during the Postweaning Period in a Porcine Model.

Exclusive breastfeeding impacts the intestinal microbiome and is associated with a better immune function than is seen with milk formula (MF) feeding in infants and yet with mechanisms poorly defined. The porcine model was used to evaluate the impact of MF on ileum microbial communities and gene expression relative to human milk (HM)-fed piglets. Fifty-two Dutch Landrace male piglets were fed an isocaloric diet of either HM (n = 26) or MF (n = 26) from day 2 through day 21 of age and weaned to a solid diet until day 51. Eleven piglets from each group were euthanized at day 21, while the remaining piglets (HM, n = 15; MF, n = 15) were euthanized at day 51 to collect ileal epithelium (EP) scrapings and ileal (IL) tissues. The epithelial mucosa was subjected to shotgun metagenome sequencing, and EP and IL tissues were used for transcriptome analysis. On day 21, transcriptome data revealed that the levels of pathways involved in inflammation and apoptosis were significantly higher in MF piglets than in HM piglets, whereas the levels of tight junctions and pathogen detection systems were lower in MF piglets than in HM piglets. The MF impacts on the small intestine were maintained over the postweaning period (day 51) as indicated by higher levels of Dialister invisus bacteria and higher levels of expression of genes associated with inflammation and apoptosis pathways relative to HM group. The current study demonstrated that MF might impact local intestinal inflammation, apoptosis, and tight junctions and might suppress pathogen recognition in the small intestine compared with HM.IMPORTANCE Exclusive human milk (HM) breastfeeding for the first 6 months of age in infants is recommended to improve health outcomes during early life and beyond. When women are unable to provide sufficient HM, milk formula (MF) is often recommended as a complementary or alternative source of nutrition. Previous studies in piglets demonstrated that MF alters the gut microbiome and induces inflammatory cytokine production. The links between MF feeding, gut microbiome, and inflammation status are unclear due to challenges associated with the collection of intestinal samples from human infants. The current report provides the first insight into MF-microbiome-inflammation connections in the small intestine compared with HM feeding using a porcine model. The present results showed that, compared with HM, MF might impact immune function through the induction of ileal inflammation, apoptosis, and tight junction disruptions and likely compromised immune defense against pathogen detection in the small intestine relative to piglets that were fed HM.

Neonatal Diet Impacts Circulatory miRNA Profile in a Porcine Model.

microRNAs (miRNAs) are conserved non-coding small nucleotide molecules found in nearly all species and breastmilk. miRNAs present in breastmilk are very stable to freeze-thaw, RNase treatment, and low pH as they are protected inside exosomes. They are involved in regulating several physiologic and pathologic processes, including immunologic pathways, and we have demonstrated better immune response to vaccines in piglets fed with human milk (HM) in comparison to dairy-based formula (MF). To understand if neonatal diet impacts circulatory miRNA expression, serum miRNA expression was evaluated in piglets fed HM or MF while on their neonatal diet at postnatal day (PND) 21 and post-weaning to solid diet at PND 35 and 51. MF fed piglets showed increased expression of 14 miRNAs and decreased expression of 10 miRNAs, relative to HM fed piglets at PND 21. At PND 35, 9 miRNAs were downregulated in the MF compared to the HM group. At PND 51, 10 miRNAs were decreased and 17 were increased in the MF relative to HM suggesting the persistent effect of neonatal diet. miR-148 and miR-181 were decreased in MF compared to HM at PND 21. Let-7 was decreased at PND 35 while miR-199a and miR-199b were increased at PND 51 in MF compared to HM. Pathway analysis suggested that many of the miRNAs are involved in immune function. In conclusion, we observed differential expression of blood miRNAs at both PND 21 and PND 51. miRNA found in breastmilk were decreased in the serum of the MF group, suggesting that diet impacts circulating miRNA profiles at PND 21. The miRNAs continue to be altered at PND 51 suggesting a persistent effect of the neonatal diet. The sources of miRNAs in circulation need to be evaluated, as the piglets were fed the same solid diet leading up to PND 51 collections. In conclusion, the HM diet appears to have an immediate and persistent effect on the miRNA profile and likely regulates the pathways that impact the immune system and pose benefits to breastfed infants.

Endocervical miRNA Expression Profiles in Women Positive for Chlamydia trachomatis with Clinical Signs and/or Symptoms Are Distinct from Those in Women Positive for Chlamydia trachomatis without Signs and Symptoms.

Chlamydia trachomatis is the leading cause of sexually transmitted infections that may progress to pelvic inflammatory disease and infertility. No effective vaccine exists for Chlamydia, nor are there biomarkers available that readily predict disease progression. In this cross-sectional pilot study, we recruited symptomatic and asymptomatic women with C. trachomatis (CT) infection and asymptomatic, uninfected control women from an urban sexually transmitted disease clinic to determine if there were differences in microRNA (miRNA) expression. Infected women with signs and/or symptoms (CTSS) have distinct miRNA profiles compared to asymptomatic infected women (CTNS). In the CTSS group, miR-142 and -147 showed 2.2- to 6.9-fold increases in expression. In the CTNS group, miR-449c, -6779, -519d, -449a, and -2467 showed 3.9- to 9.0-fold increases in expression. In the CTNS group, cyclins and cell cycle regulation and IL-17 pathways were likely downregulated, while the same signaling pathways were upregulated in the CTSS group. In addition, in the CTSS group, additional inflammatory pathways associated with TNFR1 and IL-8 appear to be upregulated. The miRNA expression patterns differ between CT-infected symptomatic and asymptomatic women, and these differences may warrant further study.

ProteoViz: a tool for the analysis and interactive visualization of phosphoproteomics data.

Quantitative proteomics generates large datasets with increasing depth and quantitative information. With the advance of mass spectrometry and increasingly larger data sets, streamlined methodologies and tools for analysis and visualization of phosphoproteomics are needed both at the protein and modified peptide levels. To assist in addressing this need, we developed ProteoViz, which includes a set of R scripts that perform normalization and differential expression analysis of both the proteins and enriched phosphorylated peptides, and identify sequence motifs, kinases, and gene set enrichment pathways. The tool generates interactive visualization plots that allow users to interact with the phosphoproteomics results and quickly identify proteins and phosphorylated peptides of interest for their biological study. The tool also links significant phosphosites with sequence motifs and pathways that will help explain the experimental conditions and guide future experiments. Here, we present the workflow and demonstrate its functionality by analyzing a phosphoproteomic data set from two lymphoma cell lines treated with kinase inhibitors. The scripts and data are freely available at and via the ProteomeXchange with identifier PXD015606.

Exploiting Correlations between Protein Abundance and the Functional Status of saeRS and sarA To Identify Virulence Factors of Potential Importance in the Pathogenesis of Staphylococcus aureus Osteomyelitis.

We used a murine model of postsurgical osteomyelitis (OM) to evaluate the relative virulence of the Staphylococcus aureus strain LAC and five isogenic variants that differ in the functional status of saeRS and sarA relative to each other. LAC and a variant in which saeRS activity is increased (saeC) were comparably virulent to each other, while ΔsaeRS, ΔsarA, ΔsaeRS/ΔsarA, and saeC/ΔsarA mutants were all attenuated to a comparable degree. Phenotypic comparisons including a mass-based proteomics approach that allowed us to assess the number and abundance of full-length proteins suggested that mutation of saeRS attenuates virulence in our OM model owing primarily to the decreased production of S. aureus virulence factors, while mutation of sarA does so owing to protease-mediated degradation of these same virulence factors. This was confirmed by demonstrating that eliminating protease production restored virulence to a greater extent in a LAC sarA mutant than in the isogenic saeRS mutant. Irrespective of the mechanism involved, mutation of saeRS or sarA was shown to result in reduced accumulation of virulence factors of potential importance. Thus, using our proteomics approach we correlated the abundance of specific proteins with virulence in these six strains and identified 14 proteins that were present in a significantly increased amount (log2 ≥ 5.0) in both virulent strains by comparison to all four attenuated strains. We examined biofilm formation and virulence in our OM model using a LAC mutant unable to produce one of these 14 proteins, specifically staphylocoagulase. The results confirmed that mutation of coa limits biofilm formation and, to a lesser extent, virulence in our OM model, although in both cases the limitation was reduced by comparison to the isogenic sarA mutant.

Proteomic measures of gamma oscillations.

BACKGROUND: Gamma oscillations serve complex processes, and the first stage of their generation is the reticular activating system (RAS), which mediates the gamma-activity states of waking and paradoxical sleep. We studied whether the pedunculopontine nucleus (PPN), part of the RAS in which every cell manifests intrinsic gamma oscillations, undergoes changes resulting in distinctive protein expression. NEW METHOD: We previously found that a histone deacetylation inhibitor, trichostatin A (TSA), acutely (30 min) blocked these oscillations. We developed a proteomic method for sampling stimulated and unstimulated PPN and determining protein expression in 1 mm punches of tissue from brain slices subjected to various treatments. RESULTS: We compared brain slices exposed for 30 min to TSA (unstimulated), to the cholinergic agonist carbachol (CAR), known to induce PPN gamma oscillations, or exposed to both TSA + CAR.Comparison with existing methods: Label-free proteomics provides an unbiased and sensitive method to detect protein changes in the PPN. Our approach is superior to antibody-based methods that can lack specificity and can only be done for known targets. Proteomics methods like these have been leveraged to study molecular pathways in numerous systems and disease states. CONCLUSIONS: Significant protein changes were seen in two functions essential to the physiology of the PPN: cytoskeletal and intracellular [Ca2+] regulation proteins. TSA decreased, while CAR increased, and TSA + CAR had intermediate effects, on expression of these proteins. These results support the feasibility of the methods developed for determining proteomic changes in small samples of tissue participating in the most complex of brain processes.

Continuous Developmental and Early Life Trichloroethylene Exposure Promoted DNA Methylation Alterations in Polycomb Protein Binding Sites in Effector/Memory CD4+ T Cells.

Trichloroethylene (TCE) is an industrial solvent and drinking water pollutant associated with CD4+ T cell-mediated autoimmunity. In our mouse model, discontinuation of TCE exposure during adulthood after developmental exposure did not prevent immunotoxicity. To determine whether persistent effects were linked to epigenetic changes we conducted whole genome reduced representation bisulfite sequencing (RRBS) to evaluate methylation of CpG sites in autosomal chromosomes in activated effector/memory CD4+ T cells. Female MRL+/+ mice were exposed to vehicle control or TCE in the drinking water from gestation until ~37 weeks of age [postnatal day (PND) 259]. In a subset of mice, TCE exposure was discontinued at ~22 weeks of age (PND 154). At PND 259, RRBS assessment revealed more global methylation changes in the continuous exposure group vs. the discontinuous exposure group. A majority of the differentially methylated CpG regions (DMRs) across promoters, islands, and regulatory elements were hypermethylated (~90%). However, continuous developmental TCE exposure altered the methylation of 274 CpG sites in promoters and CpG islands. In contrast, only 4 CpG island regions were differentially methylated (hypermethylated) in the discontinuous group. Interestingly, 2 of these 4 sites were also hypermethylated in the continuous exposure group, and both of these island regions are associated with lysine 27 on histone H3 (H3K27) involved in polycomb complex-dependent transcriptional repression via H3K27 tri-methylation. CpG sites were overlapped with the Open Regulatory Annotation database. Unlike the discontinuous group, continuous TCE treatment resulted in 129 DMRs including 12 unique transcription factors and regulatory elements; 80% of which were enriched for one or more polycomb group (PcG) protein binding regions (i.e., SUZ12, EZH2, JARID2, and MTF2). Pathway analysis of the DMRs indicated that TCE primarily altered the methylation of genes associated with regulation of cellular metabolism and cell signaling. The results demonstrated that continuous developmental exposure to TCE differentially methylated binding sites of PcG proteins in effector/memory CD4+ cells. There were minimal yet potentially biologically significant effects that occurred when exposure was discontinued. These results point toward a novel mechanism by which chronic developmental TCE exposure may alter terminally differentiated CD4+ T cell function in adulthood.