Physiological Genomics

Inorganic arsenic (iAs) in drinking water is a global health concern. This study tests whether maternal exposure to iAs in drinking water at the WHO provisional level (10{micro}g/L) increases offspring asthma risk via epigenetic reprogramming. F1 mice prenatally exposed to iAs were analyzed at 5 months for blood transcriptome and methylome changes and challenged with house allergens before lung function testing. Prenatal iAs exposure led to increased airway hyperresponsiveness (AHR) and altered inflammation gene expression and DNA methylation changes. Notably, miR-101c was epigenetically reprogrammed early in development, with persistent downregulation in both target (fetal and adult lungs) and surrogate (amniotic fluid and blood) tissues. These changes correlated with increased allergic AHR and TGF{beta} pathway dysregulation. Findings suggest that maternal iAs exposure primes offspring for asthma risk through epigenetic alterations and may inform risk assessment and biomarker development in affected communities. KEY FINDINGSO_LIIn utero exposure to 10 part per billion (or 10 {micro}g/L, the current WHO and EPA provisional level in drinking water) inorganic arsenic (iAs) increases offspring asthma risk. These results raise concerns about the current safety thresholds for iAs in drinking water (Fig. 1). C_LIO_LITranscriptomic and methylome analyses of blood leukocytes from 5-month-old F1 mice revealed that maternal iAs exposure induces transcriptional changes in genes related to allergic airway responses. Pathway analysis highlighted the involvement of miR-101c and its connection with TGF{beta} downstream targets in regulating extracellular matrix signaling, embryonic development, and inflammatory (Figs. 2 and 3). C_LIO_LITranscriptional changes in col3a1 and miR-101c in blood were strongly correlated with allergen-induced airway hyperresponsiveness (AHR), with similar alterations detected in plasma samples. These findings provide new insights into respiratory health in affected communities and support the development of biomarkers for iAs risk assessment (Figs. 4 and 5). C_LIO_LISeveral gene-specific epigenetic alterations induced by early-life iAs exposure were consistently observed in both surrogate (blood) and target (lung) tissues across developmental stages, offering new panels of easily accessible markers for early detection and monitoring of lung disease risk in offspring. The persistence of these markers over time makes them valuable for predictive modeling and life course studies. (Fig. 6). C_LIO_LIDownregulation of miR-101c was validated in fetal lung and amniotic fluid of the iAs-exposed group, suggesting that epigenetic reprogramming of miR-101c is initiated early in gestation (Fig. 7). These findings help uncover causal pathways linking environmental exposures to asthma pathogenesis. C_LIO_LIDistinct sex-specific patterns in blood transcriptome and methylome alterations in respond to early-life iAs exposure underscore the importance of considering sex as a biological variable in omics research. C_LI O_FIG O_LINKSMALLFIG WIDTH=149 HEIGHT=200 SRC="FIGDIR/small/668223v1_fig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1f5394eorg.highwire.dtl.DTLVardef@855bf8org.highwire.dtl.DTLVardef@1592328org.highwire.dtl.DTLVardef@1133ec4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 1.C_FLOATNO Maternal iAs exposure and study design to assess offspring asthma risk. A) Schematic shows the exposure window in which F0 dams received iAs in drinking water from preconception through lactation, followed by collection of F1 progeny for transcriptomic and methylomic analysis. A subset of 5-month-old F1 progenies were assessed for the HDM allergen-induced airway reactivity and airway inflammation. B) Lung resistance was assessed following methacholine (30mg/mL) challenge. C) BAL cells were stained with Diff-Quick, and eosinophil counts are shown. Each datapoint represents a pup from an individual dam; error bars indicate SEM. Five to six dams were used. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/668223v1_fig2.gif" ALT="Figure 2"> View larger version (23K): org.highwire.dtl.DTLVardef@7dc1b1org.highwire.dtl.DTLVardef@378529org.highwire.dtl.DTLVardef@1076fe3org.highwire.dtl.DTLVardef@142d801_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 2.C_FLOATNO Blood transcriptome profiles and predicted canonical pathways and regulatory networks. A) Numbers of differentially expressed genes (DEGs) identified in male and female progeny are illustrated with a Venn diagram. Enriched canonical pathways ranked by the p value are shown. B) Using the set of DEGs common in both sexes, Ingenuity Pathway Analysis (IPA) highlights TGF as a central upstream regulator linking overlapping DEGs related to extracellular matrix signaling (orange), inflammation (yellow), and embryonic development/cell metabolism (blue). C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/668223v1_fig3.gif" ALT="Figure 3"> View larger version (23K): org.highwire.dtl.DTLVardef@1732b53org.highwire.dtl.DTLVardef@216332org.highwire.dtl.DTLVardef@1f794d1org.highwire.dtl.DTLVardef@14b62df_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 3.C_FLOATNO Blood methylome analysis and associations between miR-101c and TGF-related differentially expressed genes. A) Number of differentially methylated CpGs (DMCs) identified in male and female progeny are illustrated with a Venn diagram. There are four DMCs overlapping DMCs annotated to miR-101c. B) Expression levels of miR-101c are shown. C) IPA-derived network linking this miRNA to TGF-related DEGs. Each datapoint represents a pup from an individual dam; error bars indicate SEM. Four to five dams were used. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/668223v1_fig4.gif" ALT="Figure 4"> View larger version (38K): org.highwire.dtl.DTLVardef@482e86org.highwire.dtl.DTLVardef@e666bdorg.highwire.dtl.DTLVardef@631538org.highwire.dtl.DTLVardef@10618f6_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 4.C_FLOATNO Relationship between iAs-induced blood transcriptional changes and allergen-induced AHR. This figure presents correlation patterns between baseline blood DEGs expression changes prior to HDM challenge and subsequent changes in AHR. Pearson correlation coefficients are depicted using a color gradient to distinguish positive (blue) and negative (peach) associations. * Positive correlation: r>0.8, p<0.05; ^ negative correlation: r<-0.8, p<0.05 C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/668223v1_fig5.gif" ALT="Figure 5"> View larger version (20K): org.highwire.dtl.DTLVardef@150d27eorg.highwire.dtl.DTLVardef@12b4edeorg.highwire.dtl.DTLVardef@4702a8org.highwire.dtl.DTLVardef@1ff9600_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 5.C_FLOATNO Circulating miR-101c levels in progeny exposed to maternal iAs. Plasma levels of miR-101c and COL3A1 protein are shown at different developmental stages. A) Relative expression of miR-101c across F1 progeny at juvenile (3 weeks of age) and adult (5 months of age) prenatally exposed to iAs. B) Levels of COL3A1 protein are shown at juvenile and adult F1 progeny. Each datapoint represents a pup from an individual dam, error bars indicate SEM. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=160 SRC="FIGDIR/small/668223v1_fig6.gif" ALT="Figure 6"> View larger version (46K): org.highwire.dtl.DTLVardef@d0ec76org.highwire.dtl.DTLVardef@5468a2org.highwire.dtl.DTLVardef@1a17e85org.highwire.dtl.DTLVardef@17956ce_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 6.C_FLOATNO Comparison of transcriptional patterns across tissues and developmental stages. Expression changes (FC: fold changes vs. unexposed) of DEGs and miR-101c are compared between blood and lung tissues in both A) juvenile (3 weeks of age) and B) adult (5 months of age) progeny. Blue and orange bars represent FC in blood from male and female progeny, respectively. Black bars represent FC in lung tissues. Solid bars indicate statistically significant changes. C_FIG O_FIG O_LINKSMALLFIG WIDTH=154 HEIGHT=200 SRC="FIGDIR/small/668223v1_fig7.gif" ALT="Figure 7"> View larger version (22K): org.highwire.dtl.DTLVardef@131ef23org.highwire.dtl.DTLVardef@14b0652org.highwire.dtl.DTLVardef@8c1b30org.highwire.dtl.DTLVardef@4cf6d9_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 7.C_FLOATNO Fetal lung and amniotic fluid levels of miR-101c. Relative levels of miR-101c are shown in fetal lung tissues and matched amniotic fluid at embryonic day 16. Data illustrate expression patterns in the context of maternal iAs exposure. Each datapoint represents a pup from an individual dam, error bars indicate SEM. C_FIG GRAPHIC ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/668223v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@e25f49org.highwire.dtl.DTLVardef@1dddc22org.highwire.dtl.DTLVardef@9f4244org.highwire.dtl.DTLVardef@1b787e9_HPS_FORMAT_FIGEXP M_FIG C_FIG Description: F1 progeny prenatally exposed to iAs were assessed for blood transcriptomic and DNA methylome analysis at 5 months of age. Correlation analysis between transcriptional changes and allergen-induced airway hyperresponsiveness (AHR) was conducted to examine the epigenetic impact of maternal iAs exposure on offspring asthma risk.

C57BL/6J and BALB/cJ mouse strains were analyzed by deep mRNA sequencing of the liver in the fasted state and following ingestion of standard laboratory mouse chow supplemented with plain drinking water or water containing 20% glucose, sucrose or fructose. Supplementation with these carbohydrates induced unique extents and temporal changes in gene expressions in a strain specific manner. Fructose and sucrose stimulated gene changes peaked at 3 h postprandial, whereas glucose effects peaked at 12 h postprandial in C57BL/6J mice and at 6 h postprandial in BABL/cJ mice. Network analyses revealed that fructose changed genes were primarily involved in lipid metabolism and were more complex in C57BL/6J than in BALB/cJ mice. These data demonstrate that there are qualitative and quantitative differences in the normal physiological responses of the liver between these two strains of mice and C57BL/6J is more sensitive to sugar intake than BALB/cJ.

Understanding how heterogeneous {beta}-cell function and stress response impact diabetic etiology is imperative for therapy development. Standard single-cell RNA sequencing analysis illuminates some genetic underpinnings driving heterogeneity, but new strategies are required to capture information lost due to technical limitations. Here, we integrate pancreatic islet single-cell and bulk RNA sequencing data to identify {beta}-cell subpopulations based on gene expression and characterize genetic networks associated with {beta}-cell function in high- and low-fat fed male and female SM/J mice at 20 and 30wks of age. Previous studies have shown that high-fat fed SM/J mice resolve glycemic dysfunction between 20 and 30wks. We identify 4 {beta}-cell subpopulations associated with insulin secretion, hypoxia response, cell polarity, and stress response. Relative proportions of these cells are influenced by age, sex, and diet. Network analysis identifies fatty acid metabolism and {beta}-cell physiology gene expression modules associated with the hyperglycemic-obese state. We identify subtype-specific expression of Pdyn and Fam151a as candidate regulators of genetic pathways associated with {beta}-cell function in obesity. In sum, this study uses a novel data integration method to explore how {beta}-cells respond to obesity and glycemic stress, helping to define the relationship between {beta}-cell heterogeneity and diabetes, and shedding light on novel genetic pathways with therapeutic potential.

A data-independent acquisition (DIA) assay library was generated for the liver of threespine sticklebacks to evaluate alterations in protein abundance and functional enrichment of molecular pathways following either chronic warm (25{degrees}C) or cold (7{degrees}C) three-week temperature challenge in two estuarine populations. The DIA assay library was created from a data-dependent acquisition (DDA) based raw spectral library that was filtered to remove low quality or ambiguous peptides. Functional enrichment analyses using STRING identified larger networks that were significantly enriched by examining both the entire liver proteome and only significantly elevated or depleted proteins from the various comparisons. These systems level analyses revealed the unique liver proteomic signatures of two populations of threespine sticklebacks acclimated to chronic temperature stress. The Big lagoon population (BL) had a stronger response than the Klamath river population (KL). At 7{degrees}C, BL showed alterations in protein homeostasis that likely fueled a higher demand for energy, but both populations successfully acclimated to this temperature. The warm acclimation induced major increases in proteins involved in chromatin structure and transcription, while there were decreases in proteins related to translation and fatty acid metabolism. Functional enrichment analyses of the entire liver proteome uncovered differences in glycolysis and carbohydrate metabolism between the two populations and between the cold acclimated and control groups. We conclude that the synchronous regulatory patterns of many proteins observed in the liver of threespine sticklebacks provide more comprehensive insight into population-specific responses to thermal stress than the use of less specific pre-determined biomarkers.

BackgroundGenomic imprinting is an epigenetic phenomenon that results in parent-of-origin-specific gene expression and has been extensively characterized in mice and humans. However, in pigs, imprinting has been investigated primarily through analyses of orthologs of known imprinted genes in mice and humans. The objective of this study was to examine DNA methylation status and gene expression at a porcine locus containing newly identified imprinted calcitonin receptor-stimulating peptide (CRSP)-encoding genes, to compare orthologous loci in mice and humans, and to investigate a potential underlying mechanism. ResultsAnalyses of differentially methylated regions (DMRs) between porcine parthenogenetic embryos and biparental controls revealed multiple parental DMRs at a locus we term the CRSP complex locus, which harbors CRSP-encoding genes that likely arose through gene duplication. In contrast, orthologous genomic intervals in mice and humans exhibited unmethylated promoters and lacked evidence of imprinting. Consistently, CRSP-encoding genes in pigs showed parent-of-origin-specific monoallelic expression, whereas genes within the orthologous locus in mice and humans were biallelically expressed. Further analysis indicated that porcine CRSP promoters are embedded within oocyte-expressed alternative transcripts and co-occurred with DNA methylation, suggesting a transcription-dependent imprinting mechanism. ConclusionsOur comparative analyses identified CRSP-encoding genes at the porcine CRSP complex locus as novel imprinted genes, indicating species-specific evolution of this imprinted domain. The results further suggest that lineage-specific gene duplication may have contributed to the emergence of imprinting at this locus.

Glucocorticoids (GCs) are widely used anti-inflammatory and immunosuppressive agents known to induce dramatic changes in gene expression, yet their effects on non-coding RNAs such as long non-coding RNAs (lncRNAs) or microRNAs (miRNAs) remain poorly characterized. We present the first comprehensive and systematic analysis of GC regulation of the non-coding genome across nine human primary cell types from both hematopoietic and non-hematopoietic lineages. In vitro GC treatment was applied to each cell type, with transcriptomic profiling (total RNA-seq and small RNA-seq) at 2 and 6 hours post-treatment. We identified over 2,000 GC-responsive non-coding transcripts, including 654 annotated lncRNAs, 1,376 novel lncRNAs, and 39 miRNAs. The non-coding RNA response to GCs was highly cell type-dependent: 80% of GC-responsive lncRNAs and 97% of miRNAs were unique to a single cell type. Hematopoietic cells exhibited a greater magnitude of lncRNA induction than non-hematopoietic cells. Notably, dozens of facultative lncRNAs (undetectable at baseline) were induced de novo by GC. GC-responsive transcripts spanned diverse lncRNA classes, with enrichment of host lncRNAs. By contrast, only a limited number of miRNAs were GC-responsive. GC regulation of transcript abundance for miRNAs and their host lncRNAs appears to be independent. Our results, which are accessible through an interactive web application, establish a framework for studying how non-coding transcription contributes to physiological and clinical heterogeneity in GC responses. The newly identified GC-responsive non-coding transcripts could represent biomarkers of GC exposure, determinants of GC sensitivity or resistance, or candidate regulators of tissue-specific GC effects.

BackgroundPoor nutrition during development programs kidney function. No studies on postnatal consequences of decreased perinatal nutrition exist in nonhuman primates (NHP) for translation to human renal disease. Our baboon model of moderate maternal nutrient restriction (MNR) produces intrauterine growth restricted (IUGR) and programs renal fetal phenotype. We hypothesized that the IUGR phenotype persists postnatally, influencing responses to a high-fat, high-carbohydrate, high-salt (HFCS) diet. MethodsPregnant baboons ate chow (Control; CON) or 70% of control intake (MNR) from 0.16 gestation through lactation. MNR offspring were IUGR at birth. At weaning, all offspring (CON and IUGR females and males, n=3/group) ate chow. At ~4.5 years of age, blood, urine, and kidney biopsies were collected before and after a 7-week HFCS diet challenge. Kidney function, unbiased kidney gene expression, and untargeted urine metabolomics were evaluated. ResultsIUGR female and male kidney transcriptome and urine metabolome differed from CON at 3.5 years, prior to HFCS. After the challenge, we observed sex-specific and fetal exposure-specific responses in urine creatinine, urine metabolites, and renal signaling pathways. ConclusionsWe previously showed mTOR signaling dysregulation in IUGR fetal kidneys. Before HFCS, gene expression analysis indicated that dysregulation persists postnatally in IUGR females. IUGR male offspring response to HFCS showed uncoordinated signaling pathway responses suggestive of proximal tubule injury. To our knowledge, this is the first study comparing CON and IUGR postnatal juvenile NHP and the impact of fetal and postnatal life caloric mismatch. Perinatal history needs to be taken into account when assessing renal disease risk.

The contribution of microRNAs (miRNAs) to mRNA regulation has often been explored by post hoc selection of downregulated genes and determining whether they harbor binding sites for miRNAs of interest. This approach, however, does not discriminate whether these mRNAs are also downregulated at the transcriptional level. Here, we have characterized the transcriptional and post-transcriptional changes of mRNA expression in two porcine tissues: gluteus medius muscle of fasted and fed Duroc gilts and adipose tissue of lean and obese Duroc-Gottingen minipigs. Exon-intron split analysis (EISA) of RNA-seq data allowed us to identify downregulated mRNAs with high post-transcriptional signals in fed or obese states, and we assessed whether they harbor binding sites for upregulated miRNAs in any of these two physiological states. We found 26 downregulated mRNAs with high post-transcriptional signals in the muscle of fed gilts and 21 of these were predicted targets of upregulated miRNAs also in the fed state. For adipose tissue, 44 downregulated mRNAs in obese minipigs displayed high post-transcriptional signals, and 25 of these were predicted targets of miRNAs upregulated in the obese state. These results suggest that the contribution of miRNAs to mRNA repression is more prominent in the skeletal muscle system. Finally, we identified several genes that may play relevant roles in the energy homeostasis of the pig skeletal muscle (DKK2 and PDK4) and adipose (SESN3 and ESRRG) tissues. By differentiating transcriptional from post-transcriptional changes in mRNA expression, EISA provides a valuable view about the regulation of gene expression, complementary to canonical differential expression analyses.

BackgroundNeurodevelopmental disorders have a strong male bias that is poorly understood. Placenta is a rich source of molecular information about environmental interactions with genetics (including biological sex), that affect the developing brain. We investigated placental-brain transcriptional responses in an established mouse model of prenatal exposure to a human-relevant mixture of polychlorinated biphenyls (PCBs). ResultsTo understand sex, tissue, and dosage effects in embryonic (E18) brain and placenta by RNAseq, we used weighted gene correlation network analysis (WGCNA) to create correlated gene networks that could be compared across sex or tissue. WGCNA revealed that expression within most correlated gene networks was significantly and strongly associated with PCB exposures, but frequently in opposite directions between male-female and placenta-brain comparisons. In both WGCNA and differentially expressed gene analyses, male brain showed more PCB-induced transcriptional changes than male placenta, but the reverse pattern was seen in females. Furthermore, non-monotonic dose responses to PCBs were observed in most gene networks but were most prominent in male brain. The transcriptomic effects of low dose PCB exposure were significantly reversed by dietary folic acid supplementation across both sexes, but these effects were strongest in female placenta. PCB-dysregulated and folic acid-reversed gene networks were commonly enriched in functions in metabolic pathways involved in energy usage and translation, with female-specific protective effects enriched in PPAR, thermogenesis, glycerolipids, and O-glycan biosynthesis, as opposed to toxicant responses in male brain. ConclusionsThe female protective effect in prenatal PCB exposures appears to be mediated by dose-dependent sex differences in transcriptional modulation of metabolism in placenta. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/603326v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@14d4936org.highwire.dtl.DTLVardef@101b87eorg.highwire.dtl.DTLVardef@1f787e6org.highwire.dtl.DTLVardef@1fb1b62_HPS_FORMAT_FIGEXP M_FIG C_FIG

Host associated microbiota play essential roles in regulating digestion, nutrient acquisition, immunity, and xenobiotic metabolism. Disruption of these communities is linked to numerous diseases and health defects though causal mechanisms underpinning these associations remain unclear in most cases. Gnotobiotic zebrafish provide a scalable low-cost in vivo model that is increasingly used to resolve causality in host-microbiota interactions. However, reliance on live diets limits the use of gnotobiotic zebrafish to early life stages where body systems and microbial communities are incompletely developed. As a result, many important host-microbiota interactions may be unable to be studied in this model system. Here we tested a simple method for long-term husbandry of gnotobiotic zebrafish using gamma-irradiated chow diets and evaluated effects on growth, gene expression, and microbial community composition. In conventionally reared animals, gamma irradiated diets did not affect growth or survival and only modestly impacted microbial community composition and diversity. In contrast, gnotobiotic zebrafish maintained on sterile irradiated diets for 55 days post fertilization were smaller, weighed less, and exhibited aberrant genes expression profiles relative to controls. These genes were enriched for pathways, related to immune response, xenobiotic metabolism, organ development, liver function, and lipid metabolism, with many expression patterns linked to the abundance of specific microbial taxa. Together, these findings establish a practical protocol for long-term maintenance of gnotobiotic zebrafish and extend the utility of this model to study microbiome-dependent effects on host physiology, and development beyond early larval stages of life. IMPORTANCEWhile the gnotobiotic zebrafish have been a powerful model for interrogation of host-microbiota interactions, their use has been limited to early life stages due to complications of long-term husbandry. To address this limitation, we developed a simple protocol that enables rearing germ-free zebrafish well beyond larval stages. Germ free fish exhibit physiological and developmental defects that mirror those described in mammalian counterparts supporting a conserved role for microbiota in vertebrate development and physiology. Our protocol provides a method to investigate microbial influences on adaptive immunity, metabolism, and chronic disease processes in zebrafish not possible with current methodologies. Given the rapid and simple methods for gnotobiotic derivation and the large number of transgenic animal lines available for zebrafish we anticipate this model will accelerate mechanistic discovery of microbial impacts on host health.

PurposeA growing body of evidence suggests a protective role for high-density lipoprotein (HDL) in the development and progression of sepsis. However, evidence favoring causality between HDL and sepsis remains limited. Here we investigated, by using Mendelian randomization (MR), the potential causal link between HDL particle concentration or HDL-related proteins, and the risk of developing sepsis. MethodsTwo-sample MR analyses were conducted to investigate association between genetically-influenced HDL particle concentration, HDL-related proteins, and the risk of sepsis. We utilized a genome-wide association study (GWAS) comprising 10,154 sepsis cases and 452,764 controls as outcome. Inverse-variance weighted (IVW) analyses were employed as primary analyses. Protein-protein interaction (PPI) network analyses were used to identify key regulators for concentration of HDL particles and ApoA1. ResultsBased on the IVW analyses, we found evidence for an association between genetically-influenced higher HDL particle concentration and lower risk of sepsis (odds ratio (OR): 0.86, 95% confidential interval (CI): 0.77-0.96). Similarly, genetically-influenced higher ApoA1 concentration was associated with a lower risk of sepsis (OR: 0.90, 95% CI: 0.85 - 0.96). Through PPI network analyses, we identified cholesteryl ester transfer protein (CETP) as one of the key players in regulating the concentration of HDL particles and ApoA1, and sepsis risk as such. ConclusionOur MR study provides evidence for an inverse relationship between HDL particle concentration or ApoA1, and the susceptibility to sepsis. We anticipate that CETP inhibitory strategies may increase HDL particle concentration and apoA1 to lower sepsis susceptibility and potentially prevent the risks associated with sepsis.

BackgroundGlobal and site-specific changes in DNA methylation and gene expression are associated with cardiovascular aging and disease, but how toxicant exposures during early development influence the normal trajectory of these age-related molecular changes, and whether there are sex differences, has not yet been investigated. ObjectivesWe used an established mouse model of developmental exposures to investigate the effects of perinatal exposure to either lead (Pb) or diethylhexyl phthalate (DEHP), two ubiquitous environmental contaminants strongly associated with CVD, on age-related cardiac DNA methylation and gene expression. MethodsDams were randomly assigned to receive human physiologically relevant levels of Pb (32 ppm in water), DEHP (25 mg/kg chow), or control water and chow. Exposures started two weeks prior to mating and continued until weaning at postnatal day 21 (3 weeks of age). Approximately one male and one female offspring per litter were followed to 3 weeks, 5 months, or 10 months of age, at which time whole hearts were collected (n [&ge;] 5 per sex per exposure). Enhanced reduced representation bisulfite sequencing (ERRBS) was used to assess the cardiac DNA methylome at 3 weeks and 10 months, and RNA-seq was conducted at all 3 time points. MethylSig and edgeR were used to identify age-related differentially methylated regions (DMRs) and differentially expressed genes (DEGs), respectively, within each sex and exposure group. Cell type deconvolution of bulk RNA-seq data was conducted using the MuSiC algorithm and publicly available single cell RNA-seq data. ResultsThousands of DMRs and hundreds of DEGs were identified in control, DEHP, and Pb-exposed hearts across time between 3 weeks and 10 months of age. A closer look at the genes and pathways showing differential DNA methylation revealed that the majority were unique to each sex and exposure group. Overall, pathways governing development and differentiation were most frequently altered with age in all conditions. A small number of genes in each group showed significant changes in DNA methylation and gene expression with age, including several that were altered by both toxicants but were unchanged in control. We also observed subtle, but significant changes in the proportion of several cell types due to age, sex, and developmental exposure. DiscussionTogether these data show that perinatal Pb or DEHP exposures deflect normal age-related gene expression, DNA methylation programs, and cellular composition across the life course, long after cessation of exposure, and highlight potential biomarkers of developmental toxicant exposures. Further studies are needed to investigate how these epigenetic and transcriptional changes impact cardiovascular health across the life course.

BackgroundGene expression levels are affected by genetics and environmental effects. However, quantification of the influence of genetics and environmental effects on gene expression remains limited, especially in farm animals. Here, the relative influence of genetic and heat-related environmental variations on gene expression levels was investigated in pigs, using a backcross herd of diverse heat adaptation levels. Backcross animals were raised in either a tropical or temperate environment. Animals raised in temperate environment were subjected to an experimental heat stress at the end of their growth. ResultsWe identified 1,967 differentially expressed genes (DEGs) between pigs raised in the tropical (n = 181) and temperate (n = 180) facilities, and 472 DEGs throughout a 3 weeks experimental heat stress. Transcriptome-wide association (TWAS) study identified 139 associations between gene expression levels and thermoregulation/production traits. We detected 6,014 expression quantitative trait loci (eQTLs) associated with the expression level of 3,297 genes. Genetic variance was estimated to explain 36.3% of gene expression variance on average, and was the main source of variance for 27.7% of transcripts. Most eQTLs found are located in proximal regions (cis-eQTLs) and few within distal regions (trans-eQTLs) to their assigned genes. A trans-eQTL hotspot highlighted a hematopoietic mechanism driven by GPATCH8. An integration of GWAS and TWAS pointed to TMCO1 and ZNF184 as candidate genes for backfat thickness. ConclusionsThis study provides a better understanding of the impact of climate, heat stress and genetic influences on the pig whole blood transcriptome.

BackgroundDNA methylation is a critical epigenetic mechanism linking early developmental environment to long-term health. In humans, the extent to which toxicant-induced changes in DNA methylation in surrogate tissues, such as blood, mirror those in the target tissues is unclear. 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.\n\nObjectivesThe objective of this study was to investigate the effects of perinatal exposure to a human environmentally relevant level (32 ppm in maternal drinking water) of lead (Pb) on liver and blood DNA methylation in adult male and female mice. We hypothesized that developmental Pb exposure would lead to persistent changes in DNA methylation, and that a subset of differentially methylated loci would overlap between liver and blood.\n\nMethodsEnhanced reduced-representation bisulfite sequencing was used to assess DNA methylation in 5 month old Pb-exposed and control mice. Sex-stratified modeling of differential methylation by Pb exposure was conducted using an established bioinformatics pipeline.\n\nResultsAlthough Pb exposure ceased at 3 weeks of age, we observed thousands of stably modified, sex-specific differentially methylated regions in the blood and liver of Pb-exposed animals, including 44 genomically imprinted loci. In males, we discovered 5 sites that overlapped between blood and liver, and exhibited changes in DNA methylation in the same direction in both tissues.\n\nConclusionsThese data demonstrate that perinatal exposure to Pb induces sex-specific changes in hepatic DNA methylation in adulthood, some of which are also present in blood. Ongoing studies will provide additional exposure-specific insights, and include other epigenetic marks that will enable further refinement of the design and analysis of human studies where target tissues are inaccessible.

In the United States, cardiovascular disease is the leading cause of death, and the rate of maternal mortality remains among the highest of any industrialized nation. Maternal cardiometabolic health throughout gestation and postpartum is representative of placental health and physiology. Both proper placental functionality and placental microRNA expression are essential to successful pregnancy outcomes, and both are highly sensitive to genetic and environmental sources of variation. While placental pathologies, such as preeclampsia, are associated with maternal cardiovascular health and may contribute to the developmental programming of cardiovascular disease, the role of more subtle alterations to placental function and microRNA expression in this relationship remains poorly understood. To develop a more comprehensive understanding of how cardiometabolic health influences placental microRNA expression, and how this shapes placental functionality, we performed small RNA sequencing to investigate microRNA in the placentae from the Rhode Island Child Health Study (n=230). We modeled microRNA counts on maternal family history of cardiovascular disease using negative binomial generalized linear models, and identified microRNAs that were differential expressed (DEmiRs) at a false discovery rate (FDR) less than 0.10. Utilizing parallel mRNA sequencing data and bioinformatic target prediction software, we identified potential mRNA targets of these DEmiRs. We identified 9 DEmiRs, with predicted targets of those miRNA enriched overwhelmingly in the TGF{beta} signaling pathway but also in pathways involving cellular metabolism and immunomodulation. Overall, we identified a robust association existing between familial cardiovascular disease and placental microRNA expression which may be implicated in both placental insufficiencies and the developmental programming of cardiovascular disease.

BackgroundHeat stress (HS) poses a major challenge to the dairy industry by reducing milk production, yet its cell type-specific effects in the bovine mammary gland remain incompletely defined. In this study, we recorded production traits and collected mammary biopsies from cows under thermoneutral (TN), HS, and pair-fed (PF) conditions. ResultsClinical measurements confirmed HS-induced physiological alterations. Compared with TN cows, HS cows exhibited reduced dry matter intake (DMI), milk yield, and yields of fat, protein, and lactose, along with increased water intake and milk urea nitrogen. The use of PF controls indicated that decreased DMI accounted for 45% of the milk-yield reduction, whereas direct HS effects accounted for the remaining reduction. We applied single-nucleus RNA-seq (snRNA-seq) on mammary biopsies to generate cell-resolved HS responses. We identified 14 distinct cell clusters, including epithelial, immune, and stromal populations. Under the TN condition, casein genes (e.g., CSN1S1, CSN2) were broadly expressed across luminal cells but were attenuated under HS, whereas luminal alveolar cells showed relative upregulation. Heat shock protein genes were strongly induced by HS, primarily in epithelial clusters. Gene-set enrichment analyses revealed increased ribosomal activities across HS-responsive clusters and enrichment of protein folding and metabolic pathways in luminal alveolar cells, suggesting elevated proteostasis demands under stress. Pseudotime analysis positioned luminal cells along a progenitor-to-secretory trajectory under TN, accompanied by increased casein gene expression, whereas under HS, mature luminal cells shifted toward a homeostasis regulatory state. Cell-cell communication analysis demonstrated HS-induced remodeling of interepithelial signaling, including altered ERBB4-mediated signaling from luminal hormone-sensing to alveolar lineages. Finally, transcription factor activity profiling highlighted cell type-specific HS-activated regulators and their downstream target genes. ConclusionsTogether, this cell type-resolved atlas delineates how HS alters bovine mammary epithelial function, developmental state, and intercellular crosstalk. These findings point to proteostasis pressure, disrupted signaling pathways, and rewired regulatory networks as mechanistic contributors to reduced lactational performance under HS, offering insights for improving heat resilience in dairy cattle.

Transcriptomic studies of human circadian rhythm are limited in efficacy by the invasiveness of sampling. Studies of circadian dynamics should ideally use multiple blood samples drawn at regular intervals over the course of a day. This is difficult to achieve, particularly at night, without disrupting the very circadian rhythm that is being studied. We propose a method by which blood is drawn at a single initial timepoint, then cultured and repeatedly sampled over the course of a day. This method is minimally invasive to the subject. Our results demonstrate that the expression levels of circadian genes are more closely correlated between the cultured (ex vivo) cells and live (in vivo) samples than non-circadian genes, suggesting that this method can be used for effective circadian analysis.

BackgroundObesity is a pivotal trigger for a spectrum of complex metabolic disorders. By colocalizing cis-eQTLs in adipose tissues from the GTEx consortium and trait-associated SNPs for complex traits from the GWAS Catalog within 3.6 million DNase I hypersensitive sites (DHSs), we systematically identify regulatory variants and genes that exhibit cis effects, as well as potential causal variants within the context of regulatory elements. ResultsOur analysis reveals that 229,504 (26.4%) cis-eQTLs located within DHS reside densely near the transcription start sites, contrasting with those outside of DHS. We observed that genes with higher allelic imbalance have shorter transcript lengths with larger number cis-eQTLs within DHS, and such imbalance genes are predominantly linked to signaling and immune response, whereas those with lower allelic imbalance tend to be involved in metabolism. Our composite colocalization score prioritizes 5,202 DHSs that encompass both cis-eQTLs and trait-associated SNPs, targeting 2,232 protein-coding genes and 523 lncRNAs across complex traits. We highlight the lncRNA SNHG5 as a prime example; it is associated with high-density lipoprotein levels and exhibits low allelic imbalance, and is also down-regulated in adipose tissue from patients with obesity. ConclusionsOur findings underscore the critical role of regulatory context in pinpointing causal variants and refining target genes, offering rich insights into the genetic mechanisms pertinent to obesity and providing valuable resources for the diagnosis and therapeutic targeting of metabolic diseases.

Genome-wide association studies (GWASs) for bone mineral density (BMD) have identified over 1,100 associations to date. However, identifying causal genes implicated by such studies has been challenging. Recent advances in the development of transcriptome reference datasets and computational approaches such as transcriptome-wide association studies (TWASs) and expression quantitative trait loci (eQTL) colocalization have proven to be informative in identifying putatively causal genes underlying GWAS associations. Here, we used TWAS/eQTL colocalization in conjunction with transcriptomic data from the Genotype-Tissue Expression (GTEx) project to identify potentially causal genes for the largest BMD GWAS performed to date. Using this approach, we identified 512 genes as significant (Bonferroni <= 0.05) using both TWAS and eQTL colocalization. This set of genes was enriched for regulators of BMD and members of bone relevant biological processes. To investigate the significance of our findings, we selected PPP6R3, the gene with the strongest support from our analysis which was not previously implicated in the regulation of BMD, for further investigation. We observed that Ppp6r3 deletion in mice decreased BMD. In this work, we provide an updated resource of putatively causal BMD genes and demonstrate that PPP6R3 is a putatively causal BMD GWAS gene. These data increase our understanding of the genetics of BMD and provide further evidence for the utility of combined TWAS/colocalization approaches in untangling the genetics of complex traits.

Human milk is the recommended nutrient source for newborns. The mammary gland comprises multiple cell types including epithelial cells and adipocytes. The contributions of mammary adipocytes to breast milk composition and the intersections between mammary nutrient sensing and milk lipids are not fully understood. A major nutrient sensor in most tissues is the mechanistic target of rapamycin 1 (mTORC1). To assess the role of excess nutrient sensing on mammary gland structure, function, milk composition, and offspring weights, we used an Adiponectin-Cre driven Tsc1 knockout model of adipocyte mTORC1 hyperactivation. Our results show that the knockout dams have higher milk fat contributing to higher milk caloric density and heavier offspring weight during lactation. Additionally, milk of knockout dams displayed a lower percentage of saturated fatty acids, higher percentage of monounsaturated fatty acids, and a lower milk {omega}6: {omega}3 ratio driven by increases in docosahexaenoic acid (DHA). Mammary gland gene expression analyses identified changes in eicosanoid metabolism, adaptive immune function, and contractile gene expression. Together, these results suggest a novel role of adipocyte mTORC1 in mammary gland function and morphology, milk composition, and offspring growth.