Search

Abstract Understanding the combined effects of risk factors on all-cause mortality is crucial for implementing effective risk stratification and designing targeted interventions, but such combined effects are understudied. We aim to use survival-tree based machine learning models as more flexible nonparametric techniques to examine the combined effects of multiple physiological risk factors on mortality. More specifically, we (1) study the combined effects between multiple physiological factors and all-cause mortality, (2) identify the five most influential factors and visualize their combined influence on all-cause mortality, and (3) compare the mortality cut-offs with the current clinical thresholds. Data from the 1999–2014 NHANES Survey were linked to National Death Index data with follow-up through 2015 for 17,790 adults. We observed that the five most influential factors affecting mortality are the tobacco smoking biomarker cotinine, glomerular filtration rate (GFR), plasma glucose, sex, and white blood cell count. Specifically, high mortality risk is associated with being male, active smoking, low GFR, elevated plasma glucose levels, and high white blood cell count. The identified mortality-based cutoffs for these factors are mostly consistent with relevant studies and current clinical thresholds. This approach enabled us to identify important cutoffs and provide enhanced risk prediction as an important basis to inform clinical practice and develop new strategies for precision medicine.

To distinguish DNA methylation (DNAm) from cell proportion changes in whole placental villous tissue research, we developed a robust cell type-specific DNAm reference to estimate cell composition. We collated new and existing cell type DNAm profiles quantified via Illumina EPIC or 450k microarrays. To estimate cell composition, we deconvoluted whole placental samples (n = 36) with robust partial correlation based on the top 30 hyper- and hypomethylated sites identified per cell type. To test deconvolution performance, we evaluated root mean square error in predicting principal components of DNAm variation in 204 external placental samples. We analyzed DNAm profiles (n = 368,435 sites) from 12 cell types: cytotrophoblasts (n = 18), endothelial cells (n = 19), Hofbauer cells (n = 26), stromal cells (n = 21), syncytiotrophoblasts (n = 4), six lymphocyte types (n = 36), and nucleated red blood cells (n = 11). Median cell composition was consistent with placental biology: 60.9% syncytiotrophoblast, 17.3% stromal, 8.8% endothelial, 3.7% cytotrophoblast, 3.7% Hofbauer, 1.7% nucleated red blood cells, and 1.2% neutrophils. Our expanded reference outperformed an existing reference in predicting DNAm variation (PC1, 15.4% variance explained, IQR = 21.61) with cell composition estimates (mean square error of prediction: 8.62 vs. 10.79, p-value < 0.001). This cell type reference can robustly estimate cell composition from whole placental DNAm data to detect important cell types, reveal biological mechanisms, and improve causal inference.

IntroductionBreast cancer is the second most diagnosed cancer, as well as the primary cause of cancer death in women worldwide. Of the different breast cancer subtypes, triple-negative breast cancer (TNBC) is particularly aggressive and is associated with poor prognosis. Black women are two to three times more likely to be diagnosed with TNBCs than white women. Recent experimental evidence suggests that basal-like TNBCs may derive from luminal cells which acquire basal characteristics through phenotypic plasticity, a newly recognized hallmark of cancer. Whether chemical exposures can promote phenotypic plasticity in breast cells is poorly understood. MethodsTo investigate further, we developed a high-content immunocytochemistry assay using normal human breast cells to test whether chemical exposures can impact luminal/basal plasticity by unbiased quantification of keratin 14 (KRT14), a basal-myoepithelial marker; keratin 8 (KRT8), a luminal-epithelial marker; and Hoechst 33342, a DNA marker. Six cell lines established from healthy tissue from donors to the Susan G. Komen Normal Tissue Bank were exposed for 48 hours to three different concentrations (0.1μM, 1μM, and 10μM) of eight ubiquitous chemicals (arsenic, BPA, BPS, cadmium, copper, DDE, lead, and PFNA), with documented exposure disparities in US Black women, in triplicate. Automated fluorescence image quantification was performed using Cell Profiler software, and a random-forest classifier was trained to classify individual cells as KRT8 positive, KRT14 positive, or hybrid (both KRT8 and KRT14 positive) using Cell Profiler Analyst. Results and discussionResults demonstrated significant concentration-dependent increases in hybrid populations in response to BPA, BPS, DDE, and PFNA. The increase in hybrid populations expressing both KRT14 and KRT8 is indicative of a phenotypically plastic progenitor-like population in line with known theories of carcinogenesis. Furthermore, BPA, BPS, DDE, and copper produced significant increases in cell proliferation, which could be indicative of a more malignant phenotype. These results further elucidate the relationship between chemical exposure and breast phenotypic plasticity and highlight potential environmental factors that may impact TNBC risk.

A single-cell RNA-seq analysis of placental villous tissue provides a deconvolution reference atlas of fetal and maternal placental cell types, and indicates that placental cellular heterogeneity in preeclampsia might contribute to differences in bulk gene expression.

Introduction: The developing epigenome changes rapidly, potentially making it more sensitive to toxicant exposures. DNA modifications, including methylation and hydroxymethylation, are important parts of the epigenome that may be affected by environmental exposures. However, most studies do not differentiate between these two DNA modifications, possibly masking significant effects.Methods: To investigate the relationship between DNA hydroxymethylation and developmental exposure to common contaminants, a collaborative, NIEHS-sponsored consortium, TaRGET II, initiated longitudinal mouse studies of developmental exposure to human-relevant levels of the phthalate plasticizer di(2-ethylhexyl) phthalate (DEHP), and the metal lead (Pb). Exposures to 25 mg DEHP/kg of food (approximately 5 mg DEHP/kg body weight) or 32 ppm Pb-acetate in drinking water were administered to nulliparous adult female mice. Exposure began 2 weeks before breeding and continued throughout pregnancy and lactation, until offspring were 21 days old. At 5 months, perinatally exposed offspring blood and cortex tissue were collected, for a total of 25 male mice and 17 female mice (n = 5–7 per tissue and exposure). DNA was extracted and hydroxymethylation was measured using hydroxymethylated DNA immunoprecipitation sequencing (hMeDIP-seq). Differential peak and pathway analysis was conducted comparing across exposure groups, tissue types, and animal sex, using an FDR cutoff of 0.15.Results: DEHP-exposed females had two genomic regions with lower hydroxymethylation in blood and no differences in cortex hydroxymethylation. For DEHP-exposed males, ten regions in blood (six higher and four lower) and 246 regions (242 higher and four lower) and four pathways in cortex were identified. Pb-exposed females had no statistically significant differences in blood or cortex hydroxymethylation compared to controls. Pb-exposed males, however, had 385 regions (all higher) and six pathways altered in cortex, but no differential hydroxymethylation was identified in blood.Discussion: Overall, perinatal exposure to human-relevant levels of two common toxicants showed differences in adult DNA hydroxymethylation that was specific to sex, exposure type, and tissue, but male cortex was most susceptible to hydroxymethylation differences by exposure. Future assessments should focus on understanding if these findings indicate potential biomarkers of exposure or are related to functional long-term health effects.

Saliva is a widely used biological sample, especially in pediatric research, containing a heterogenous mixture of immune and epithelial cells. Associations of exposure or disease with saliva DNA methylation can be influenced by cell-type proportions. Here, we developed a saliva cell-type DNA methylation reference panel to estimate interindividual cell-type heterogeneity in whole saliva studies. Saliva was collected from 22 children (7–16 years) and sorted into immune and epithelial cells, using size exclusion filtration and magnetic bead sorting. DNA methylation was measured using the Illumina MethylationEPIC BeadChip. We assessed cell-type differences in DNA methylation profiles and tested for enriched biological pathways. Immune and epithelial cells differed at 181,577 (22.8%) DNA methylation sites (t-test p

Early developmental environment can influence long-term health through reprogramming of the epigenome. Human environmental epigenetics studies rely on surrogate tissues, such as blood, to assess the effects of environment on disease-relevant but inaccessible target tissues. However, the extent to which environment-induced epigenetic changes are conserved between these tissues is unclear. A better understanding of this conservation is imperative for effective design and interpretation of human environmental epigenetics studies. The Toxicant Exposures and Responses by Genomic and Epigenomic Regulators of Transcription (TaRGET II) consortium was established by the National Institute of Environmental Health Sciences to address the utility of surrogate tissues as proxies for toxicant-induced epigenetic changes in target tissues. We and others have recently reported that perinatal exposure to lead (Pb) is associated with adverse metabolic outcomes. Here, we investigated the sex-specific effects of perinatal exposure to a human environmentally relevant level of Pb on DNA methylation in paired liver and blood samples from adult mice using enhanced reduced-representation bisulphite sequencing. Although Pb exposure ceased at 3 weeks of age, we observed thousands of sex-specific differentially methylated cytosines in the blood and liver of Pb-exposed animals at 5 months of age, including 44 genomically imprinted loci. We observed significant tissue overlap in the genes mapping to differentially methylated cytosines. A small but significant subset of Pb-altered genes exhibit basal sex differences in gene expression in the mouse liver. Collectively, these data identify potential molecular targets for Pb-induced metabolic diseases, and inform the design of more robust human environmental epigenomics studies.

Environmental contaminants such as the metal lead (Pb) are associated with cardiovascular disease, but the underlying molecular mechanisms are poorly understood. In particular, little is known about how exposure to Pb during early development impacts the cardiac epigenome at any point across the life course and potential differences between sexes. In a mouse model of human-relevant perinatal exposures, we utilized RNA-seq and Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) to investigate the effects of Pb exposure during gestation and lactation on gene expression and DNA methylation, respectively, in the hearts of male and female mice at weaning. For ERRBS, we identified differentially methylated CpGs (DMCs) or differentially methylated 1000 bp regions (DMRs) based on a minimum absolute change in methylation of 10% and an FDR < 0.05. For gene expression data, an FDR < 0.05 was considered significant. No individual genes met the FDR cutoff for gene expression; however, we found that Pb exposure leads to significant changes in the expression of gene pathways relevant to cardiovascular development and disease. We further found that Pb promotes sex-specific changes in DNA methylation at hundreds of gene loci (280 DMCs and 99 DMRs in males, 189 DMCs and 121 DMRs in females), and pathway analysis revealed that these CpGs and regions collectively function in embryonic development. In males, differential methylation also occurred at genes related to immune function and metabolism. We then investigated whether genes exhibiting differential methylation at weaning were also differentially methylated in hearts from a cohort of Pb-exposed mice at adulthood. We found that a single gene, Galnt2, showed differential methylation in both sexes and time points. In a human cohort investigating the influence of prenatal Pb exposure on the epigenome, we also observed an inverse association between first trimester Pb concentrations and adolescent blood leukocyte DNA methylation at a locus in GALNT2, suggesting that this gene may represent a biomarker of Pb exposure across species. Together, these data, across two time points in mice and in a human birth cohort study, collectively demonstrate that Pb exposure promotes sex-specific programming of the cardiac epigenome, and provide potential mechanistic insight into how Pb causes cardiovascular disease.

Microfold cells (M-cell) are specialized cells of the intestine that sample luminal microbiota and dietary antigens. Here the authors show that epithelial non-canonical NFκB signalling, as induced by NIK, is important for M-cells maintenance, yet constitutive NIK activation is associated with gut inflammation and inflammatory bowel disease.

Background: Chemical biomarker concentrations are driven by complex interactions between chemical use patterns, exposure pathways, and toxicokinetic parameters such as biological half-lives. Criteria to differentiate legacy from current exposures are helpful for interpreting variation in age-based and time trends of chemical exposure and identifying chemicals to which children are highly exposed. A systematic approach is needed to study temporal trends for a wide range of chemicals in the US population. Objectives: Using National Health and Nutrition Examination Survey (NHANES) data on measured biomarker concentrations for 141 chemicals from 1999 to 2014, we aim to 1) understand the influence of temporal determinants, in particular time trends, biological half-lives, and restriction dates on age-based trends, 2) systematically define an age-based pattern to identify chemicals with ongoing and high exposure in children, and 3) characterize how age-based trends for six Per- and Polyfluoroalkyl Substances (PFASs) are changing over time. Methods: We performed an integrated analysis of biological half-lives and restriction dates, compared distributions of chemical biomarker concentrations by age group, and then applied a series of regression models to evaluate the linear (βage) and nonlinear (βage2) relationships between age and chemical biomarker levels. Results: For restricted chemicals, a minimum persistence of 1 year in the human body is needed to observe substantial differences between the less exposed young population and historically exposed adults. We define a metric (βage2/βage>126.9) that identifies several phthalates, brominated flame retardants, pesticides, and metals such as lead and tungsten as elevated and ongoing exposures in children. While a substantial reduction in children's exposures was reflected in PFOS and PFOA, levels of PFNA and PFHxS in children were higher in 2013–2014 compared to those in 1999–2000. Conclusions: Integrating a series of regression models with systemized stratified analyses by age group enabled us to define an age-based pattern to identify chemicals that are of higher levels in children. Keywords: Age-based exposure, Temporal trends, Biomonitoring, Environmental chemicals

Cell line authentication is critical for preventing the use of mixed or misidentified cell lines in research. Current efforts include short tandem repeat (STR) analysis and PCR-based assays to detect mixed species cultures. Using PCR analysis with mouse-specific primers, we identified contaminating mouse DNA in growth factor conditioned medium (CM) derived from the L-WRN cell line (L-WRN CM), as well as in human organoid cultures maintained in the L-WRN CM. DNA isolated from L-WRN CM matched the L-WRN cell signature by STR analysis. Organoid lines that were positive for murine DNA by PCR were further analyzed via bulk RNA-sequencing and transcripts were aligned to the human and mouse genomes. RNA analysis failed to detect mouse-specific gene expression above background levels, suggesting no viable murine cells were present in the organoid cultures. We interpret our data to show conclusive evidence that mouse cell-derived CM can be a source of contaminating murine DNA detected in human organoid cultures, even though live, transcriptionally-active murine cells are not present. Together, our findings suggest that multiple methods may be required to authenticate human organoid or cell lines and urges cautious interpretation of DNA-based PCR cell line authentication results.

Lead (Pb) is a well-known toxicant that interferes with the development of a child’s nervous and metabolic systems and increases the risk of developing diseases later in life. Although studies have investigated epigenetic effects associated with Pb exposure, knowledge of genome-wide changes with in vivo low dose perinatal Pb exposure in multiple tissues is limited. Within the Toxicant Exposures and Responses by Genomic and Epigenomic Regulators of Transcription (TaRGET II) consortium, we utilized a mouse model to investigate tissue- and sex-specific DNA methylation. Dams were assigned to control or Pb-acetate water, respectively. Exposures started 2 weeks prior to mating and continued until weaning at post-natal day 21 (PND21). Liver and blood were collected from PND21 mice, and the DNA methylome was assessed using enhanced reduced representation bisulfite sequencing (ERRBS). We identified ∼1000 perinatal Pb exposure related differentially methylated cytosines (DMCs) for each tissue- and sex-specific comparison, and hundreds of tissue- and sex-specific differentially methylated regions (DMRs). Several mouse imprinted genes were differentially methylated across both tissues in males and females. Overall, our findings demonstrate that perinatal Pb exposure can induce tissue- and sex-specific DNA methylation changes and provide information for future Pb studies in humans.

Similarities between stem cells and cancer cells have implicated mammary stem cells in breast carcinogenesis. Recent evidence suggests that normal breast stem cells exist in multiple phenotypic states: epithelial, mesenchymal, and hybrid epithelial/mesenchymal (E/M). Hybrid E/M cells in particular have been implicated in breast cancer metastasis and poor prognosis. Mounting evidence also suggests that stem cell phenotypes change throughout the life course, for example, through embryonic development and pregnancy. The goal of this study was to use single cell RNA-sequencing to quantify cell state distributions of the normal mammary (NM) gland throughout developmental stages and when perturbed into a stem-like state in vitro using conditional reprogramming (CR). Using machine learning based dataset alignment, we integrate multiple mammary gland single cell RNA-seq datasets from human and mouse, along with bulk RNA-seq data from breast tumors in the Cancer Genome Atlas (TCGA), to interrogate hybrid stem cell states in the normal mammary gland and cancer. CR of human mammary cells induces an expanded stem cell state, characterized by increased expression of embryonic stem cell associated genes. Alignment to a mouse single-cell transcriptome atlas spanning mammary gland development from in utero to adulthood revealed that NM cells align to adult mouse cells and CR cells align across the pseudotime trajectory with a stem-like population aligning to the embryonic mouse cells. Three hybrid populations emerge after CR that are rare in NM: KRT18+/KRT14+ (hybrid luminal/basal), EPCAM+/VIM+ (hybrid E/M), and a quadruple positive population, expressing all four markers. Pseudotime analysis and alignment to the mouse developmental trajectory revealed that E/M hybrids are the most developmentally immature. Analyses of single cell mouse mammary RNA-seq throughout pregnancy show that during gestation, there is an enrichment of hybrid E/M cells, suggesting that these cells play an important role in mammary morphogenesis during lactation. Finally, pseudotime analysis and alignment of TCGA breast cancer expression data revealed that breast cancer subtypes express distinct developmental signatures, with basal tumors representing the most “developmentally immature” phenotype. These results highlight phenotypic plasticity of normal mammary stem cells and provide insight into the relationship between hybrid cell populations, stemness, and cancer.

Background: Stark racial disparities in disease incidence among American women remain a persistent public health challenge. These disparities likely result from complex interactions between genetic, social, lifestyle, and environmental risk factors. The influence of environmental risk factors, such as chemical exposure, however, may be substantial and is poorly understood. Objectives: We quantitatively evaluated chemical-exposure disparities by race/ethnicity, life stage, and time in United States (US) women (n = 38,080) by using biomarker data for 143 chemicals from the National Health and Nutrition Examination Survey (NHANES) 1999–2014. Methods: We applied a series of survey-weighted, generalized linear models using data from the entire NHANES women population along with cycle and age-group stratified subpopulations. The outcome was chemical biomarker concentration, and the main predictor was race/ethnicity with adjustment for age, socioeconomic status, smoking habits, and NHANES cycle. Results: Compared to non-Hispanic White women, the highest disparities were observed for non-Hispanic Black, Mexican American, Other Hispanic, and Other Race/Multi-Racial women with higher levels of pesticides and their metabolites, including 2,5-dichlorophenol, o,p’-DDE, beta-hexachlorocyclohexane, and 2,4-dichlorophenol, along with personal care and consumer product compounds, including parabens and monoethyl phthalate, as well as several metals, such as mercury and arsenic. Moreover, for Mexican American, Other Hispanic, and non-Hispanic black women, there were several exposure disparities that persisted across age groups, such as higher 2,4- and 2,5-dichlorophenol concentrations. Exposure levels for methyl and propyl parabens, however, were the highest in non-Hispanic black compared to non-Hispanic white children with average differences exceeding 4-fold. Exposure disparities for methyl and propyl parabens are increasing over time in Other Race/Multi-Racial women while fluctuating for non-Hispanic Black, Mexican American, and Other Hispanic. Cotinine levels are among the highest in Non-Hispanic White women compared to Mexican American and Other Hispanic women with disparities plateauing and increasing, respectively. Discussion: We systematically evaluated differences in chemical exposures across women of various race/ethnic groups and across age groups and time. Our findings could help inform chemical prioritization in designing epidemiological and toxicological studies. In addition, they could help guide public health interventions to reduce environmental and health disparities across populations. Keywords: Racial disparities, Age-based trends, Temporal trends, Environmental justice, Biomonitoring, Environmental chemicals

Diet is a major route of phthalate exposure in humans due to use in food packaging materials. School lunches may be an important contributor to phthalate exposure in children and adolescents in the US because of the large amount of packaging necessary for mass-produced foods. We used 2003–2014 National Health and Nutrition Examination Survey data to study the association between school lunch consumption and urinary phthalate metabolite concentrations in children (ages 6–11 years, N = 2196) and adolescents (ages 12–19 years, N = 2314). After adjustment for other covariates, children who Always consumed school lunch had significantly elevated urinary concentrations of the following phthalate metabolites compared to levels in children who Never ate school lunch: sum of di(2‑ethylhexyl) phthalate metabolites, (28% higher, 95% confidence interval, CI: 10, 49%); mono‑(carboxy‑octyl) phthalate (MCOP; 43% higher, 95% CI: 17, 76%) and mono‑n‑butyl phthalate (18% higher, 95% CI: 3.5, 34%). We did not find statistically significant associations in adolescents, but the trend for MCOP concentrations was similar to that of children. In sensitivity analyses, associations between 24-hour recall of cafeteria food and urinary phthalate metabolites were not statistically significant, which could indicate that associations observed with Always consuming school lunch are due to residual confounding. Our findings show that children who Always eat school lunch had higher levels of exposure to some phthalates, but the source of differences in exposure need to be evaluated in additional studies. Keywords: Phthalates, Diet, Cafeteria, Exposure assessment, Plasticizers, Endocrine disruptors

Summary: During development, the mammary gland undergoes extensive remodeling driven by stem cells. Breast cancers are also hierarchically organized and driven by cancer stem cells characterized by CD44+CD24low/− or aldehyde dehydrogenase (ALDH) expression. These markers identify mesenchymal and epithelial populations both capable of tumor initiation. Less is known about these populations in non-cancerous mammary glands. From RNA sequencing, ALDH+ and ALDH−CD44+CD24− human mammary cells have epithelial-like and mesenchymal-like characteristics, respectively, with some co-expressing ALDH+ and CD44+CD24− by flow cytometry. At the single-cell level, these cells have the greatest mammosphere-forming capacity and express high levels of stemness and epithelial-to-mesenchymal transition-associated genes including ID1, SOX2, TWIST1, and ZEB2. We further identify single ALDH+ cells with a hybrid epithelial/mesenchymal phenotype that express genes associated with aggressive triple-negative breast cancers. These results highlight single-cell analyses to characterize tissue heterogeneity, even in marker-enriched populations, and identify genes and pathways that define this heterogeneity. : In this article, Colacino and colleagues use flow-cytometry-sorted populations and single-cell analyses to investigate human mammary stem cells. They discover unexpected phenotypic and functional heterogeneity at the single-cell level, including a subpopulation of ALDH+ stem cells with a hybrid epithelial/mesenchymal phenotype and triple-negative breast cancer-like gene expression pattern. Keywords: breast stem cell, single-cell RNA, epithelial, mesenchymal, hybrid, RNA-seq

Abstract Background Tissue regeneration requires a series of steps, beginning with generation of the necessary cell mass, followed by cell migration into damaged area, and ending with differentiation and integration with surrounding tissues. Temporal regulation of these steps lies at the heart of the regenerative process, yet its basis is not well understood. The ability of zebrafish to dedifferentiate mature “post-mitotic” myocytes into proliferating myoblasts that in turn regenerate lost muscle tissue provides an opportunity to probe the molecular mechanisms of regeneration. Results Following subtotal excision of adult zebrafish lateral rectus muscle, dedifferentiating residual myocytes were collected at two time points prior to cell cycle reentry and compared to uninjured muscles using RNA-seq. Functional annotation (GAGE or K-means clustering followed by GO enrichment) revealed a coordinated response encompassing epigenetic regulation of transcription, RNA processing, and DNA replication and repair, along with protein degradation and translation that would rewire the cellular proteome and metabolome. Selected candidate genes were phenotypically validated in vivo by morpholino knockdown. Rapidly induced gene products, such as the Polycomb group factors Ezh2 and Suz12a, were necessary for both efficient dedifferentiation (i.e. cell reprogramming leading to cell cycle reentry) and complete anatomic regeneration. In contrast, the late activated gene fibronectin was important for efficient anatomic muscle regeneration but not for the early step of myocyte cell cycle reentry. Conclusions Reprogramming of a “post-mitotic” myocyte into a dedifferentiated myoblast requires a complex coordinated effort that reshapes the cellular proteome and rewires metabolic pathways mediated by heritable yet nuanced epigenetic alterations and molecular switches, including transcription factors and non-coding RNAs. Our studies show that temporal regulation of gene expression is programmatically linked to distinct steps in the regeneration process, with immediate early expression driving dedifferentiation and reprogramming, and later expression facilitating anatomical regeneration.

Genomic imprinting, a phenomenon by which genes are expressed in a monoallelic, parent-of-origin-dependent fashion, is critical for normal brain development. Expression of imprinted genes is regulated via epigenetic mechanisms, including DNA methylation (5-methylcytosine, 5mC), and disruptions in imprinting can lead to disease. Early-life exposure to the endocrine disrupting chemical bisphenol A (BPA) is associated with abnormalities in brain development and behavior, as well as with disruptions in epigenetic patterning, including 5mC and DNA hydroxymethylation (5-hydroxymethylcytosine, 5hmC). Using an established mouse model of perinatal environmental exposure, the objective of this study was to examine the effects of perinatal BPA exposure on epigenetic regulation of imprinted gene expression in adult mice. Two weeks prior to mating, dams were assigned to control chow or chow containing an environmentally relevant dose (50 µg/kg) of BPA. Exposure continued until offspring were weaned at post-natal day 21, and animals were followed until 10 months of age. Expression of three imprinted genes—Pde10a, Ppp1r9a, and Kcnq1, as well as three genes encoding proteins critical for regulation of 5mC and 5hmC—Dnmt1, Tet1, and Tet2, were evaluated in the right cortex and midbrain using qRT-PCR. Perinatal BPA exposure was associated with a significant increase in adult Kcnq1 (p = 0.04) and Dnmt1 (p = 0.02) expression in the right cortex, as well as increased expression of Tet2 in the midbrain (p = 0.03). Expression of Tet2 and Kcnq1 were positively correlated in the midbrain. Analysis of 5mC and 5hmC at the Kcnq1 locus was conducted in parallel samples using standard and oxidative bisulfite conversion followed by pyrosequencing. This analysis revealed enrichment of both 5mC and 5hmC at this locus in both brain regions. No significant changes in 5mC and 5hmC at Kcnq1 were observed with perinatal BPA exposure. Together, these data suggest that perinatal BPA exposure results in altered expression of Kcnq1, Dnmt1, and Tet2 in the adult mouse brain. Further studies with larger sample sizes are necessary to understand the mechanistic basis for these changes, as well as to determine the implications they have for brain development and function.

Acute gastroenteritis (AGE) has a significant disease burden on society. Noroviruses, rotaviruses, and astroviruses are important viral causes of AGE but are relatively understudied enteric pathogens. Recent developments in novel biomimetic human models of enteric disease are opening new possibilities for studying human-specific host–microbe interactions. Human intestinal enteroids (HIE), which are epithelium-only intestinal organoids derived from stem cells isolated from human intestinal biopsy tissues, have been successfully used to culture representative norovirus, rotavirus, and astrovirus strains. Previous studies investigated host–virus interactions at the intestinal epithelial interface by individually profiling the epithelial transcriptional response to a member of each virus family by RNA sequencing (RNA-seq). Despite differences in the tissue origin, enteric virus used, and hours post infection at which RNA was collected in each data set, the uniform analysis of publicly available datasets identified a conserved epithelial response to virus infection focused around “type I interferon production” and interferon-stimulated genes. Additionally, transcriptional changes specific to only one or two of the enteric viruses were also identified. This study can guide future explorations into common and unique aspects of the host response to virus infections in the human intestinal epithelium and demonstrates the promise of comparative RNA-seq analysis, even if performed under different experimental conditions, to discover universal and virus-specific genes and pathways responsible for antiviral host defense.

Ovarian cancer is developed from a single layer of thin epithelial cells covering the surface of ovary, named human ovarian surface epithelial cells. Like all primary human cells, human ovarian surface epithelial cells have a finite life span and will go into senescence and eventually die when cultured in vitro. Immortalized human ovarian surface epithelial cells will provide an important model system with which to study ovarian cancer initiation and progression. Here, we show that silencing p53 expression with retrovirus-mediated small interfering RNA can delay the senescence and extend cell passage number, but is not sufficient to immortalize normal ovarian surface epithelial cells. Introduction of the catalytic subunit of telomerase is similarly insufficient to achieve immortalization. However, concurrent disruption of p53 expression with small interfering RNA retroviral constructs and ectopic expression of the catalytic subunit of telomerase was sufficient to induce cellular immortalization in 3 of 3 human ovarian surface epithelial cell cultures tested. The immortalzation is associated with increased telomerase activity and telomere length, and attenuated response of cell-cycle regulatory proteins to irradiation. The resultant immortal cells continued to express the same specific cytokeratins 8 and 18 as parental cells did, indicating that the epithelial characters are still maintained in the immortal cells. In addition, the immortalized cells are non-tumorigenic and nearly diploid, which is in constrast with one immortalized by SV40 T/t antigens and hTERT. As both p53 pathway dysfunction and activation of telomerase are commonly present in human ovarian cancer, these immortal cells provide an authetic cell model system for the study of the human ovarian cancer initiation, progression, differentiation and chemoprevention. [ABSTRACT FROM AUTHOR]

Abstract Transferrin receptor (TFRC) is the major mediator for iron entry into a cell. Under excessive iron conditions, TFRC is expected to be reduced to lower iron uptake and toxicity. However, the mechanism whereby TFRC expression is maintained at high levels in iron‐enriched cancer cells and the contribution of TFRC to cancer development are enigmatic. Here the work shows TFRC is induced by adenomatous polyposis coli (APC) gene loss‐driven β‐catenin activation in colorectal cancer, whereas TFRC‐mediated intratumoral iron accumulation potentiates β‐catenin signaling by directly enhancing the activity of tankyrase. Disruption of TFRC leads to a reduction of colonic iron levels and iron‐dependent tankyrase activity, which caused stabilization of axis inhibition protein 2 (AXIN2) and subsequent repression of the β‐catenin/c‐Myc/E2F Transcription Factor 1/DNA polymerase delta1 (POLD1) axis. POLD1 knockdown, iron chelation, and TFRC disruption increase DNA replication stress, DNA damage response, apoptosis, and reduce colon tumor growth. Importantly, a combination of iron chelators and DNA damaging agents increases DNA damage response and reduces colon tumor cell growth. TFRC‐mediated iron import is at the center of a novel feed‐forward loop that facilitates colonic epithelial cell survival. This discovery may provide novel strategies for colorectal cancer therapy.

Head and neck squamous cell carcinoma (HNSCC) is the eighth most commonly diagnosed cancer in the United States. The risk of developing HNSCC increases with exposure to tobacco, alcohol and infection with human papilloma virus (HPV). HPV-associated HNSCCs have a distinct risk profile and improved prognosis compared to cancers associated with tobacco and alcohol exposure. Epigenetic changes are an important mechanism in carcinogenic progression, but how these changes differ between viral- and chemical-induced cancers remains unknown. CpG methylation at 1505 CpG sites across 807 genes in 68 well-annotated HNSCC tumor samples from the University of Michigan Head and Neck SPORE patient population were quantified using the Illumina Goldengate Methylation Cancer Panel. Unsupervised hierarchical clustering based on methylation identified 6 distinct tumor clusters, which significantly differed by age, HPV status, and three year survival. Weighted linear modeling was used to identify differentially methylated genes based on epidemiological characteristics. Consistent with previous in vitro findings by our group, methylation of sites in the CCNA1 promoter was found to be higher in HPV(+) tumors, which was validated in an additional sample set of 128 tumors. After adjusting for cancer site, stage, age, gender, alcohol consumption, and smoking status, HPV status was found to be a significant predictor for DNA methylation at an additional 11 genes, including CASP8 and SYBL1. These findings provide insight into the epigenetic regulation of viral vs. chemical carcinogenesis and could provide novel targets for development of individualized therapeutic and prevention regimens based on environmental exposures.