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Omics studies frequently use samples collected during cohort studies. Conditioning on sample availability can cause selection bias if sample availability is nonrandom. Inverse probability weighting (IPW) is purported to reduce this bias. We evaluated IPW in an epigenome-wide analysis testing the association between DNA methylation (261,435 probes) and age in healthy adolescent subjects (n = 114). We simulated age and sex to be correlated with sample selection and then evaluated four conditions: complete population/no selection bias (all subjects), naïve selection bias (no adjustment), and IPW selection bias (selection bias with IPW adjustment). Assuming the complete population condition represented the "truth," we compared each condition to the complete population condition. Bias or difference in associations between age and methylation was reduced in the IPW condition versus the naïve condition. However, genomic inflation and type 1 error were higher in the IPW condition relative to the naïve condition. Postadjustment using bacon, type 1 error and inflation were similar across all conditions. Power was higher under the IPW condition compared with the naïve condition before and after inflation adjustment. IPW methods can reduce bias in genome-wide analyses. Genomic inflation is a potential concern that can be minimized using methods that adjust for inflation.

Objective Our aim was to elucidate the role of diet in type 1 diabetes (T1D) by examining combinations of nutrient intake in the progression from islet autoimmunity (IA) to T1D. Methods We measured 2457 metabolites and dietary intake at the time of seroconversion in 132 IA-positive children in the prospective Diabetes Autoimmunity Study in the Young. IA was defined as the first of two consecutive visits positive for at least one autoantibody (insulin, GAD, IA-2, or ZnT8). By December 2018, 40 children progressed to T1D. Intakes of 38 nutrients were estimated from semiquantitative food frequency questionnaires. We tested the association of each metabolite with progression to T1D using multivariable Cox regression. Nutrient patterns that best explained variation in candidate metabolites were identified using reduced rank regression (RRR), and their association with progression to T1D was tested using Cox regression adjusting for age at seroconversion and high-risk HLA genotype. Results In stepwise selection, 22 nutrients significantly predicted at least two of the 13 most significant metabolites associated with progression to T1D, and were included in RRR. A nutrient pattern corresponding to intake lower in linoleic acid, niacin, and riboflavin, and higher in total sugars, explained 18% of metabolite variability. Children scoring higher on this metabolite-related nutrient pattern at seroconversion had increased risk for progressing to T1D (HR = 3.17, 95%CI = 1.42-7.05). Conclusions Combinations of nutrient intake reflecting candidate metabolites are associated with increased risk of T1D, and may help focus dietary prevention efforts.

Background Oxylipins are formed from oxidation of omega-6 (n6) and omega-3 (n3) fatty acids (FAs). Evidence for inflammatory effects comes mostly from adults. Methods Oxylipins from n6 FA (27 n6-oxylipins) and n3 FA (12 n3-oxylipins) were measured through ultra-high-performance liquid chromatography-mass spectrometry (LC-MS/MS) in plasma from 111 children at risk of type 1 diabetes (age 1-17 years) studied longitudinally. Oxylipin precursor FAs (arachidonic acid, linoleic acid, alpha-linolenic acid, docosahexaenoic acid, eicosapentaenoic acid) were measured in red blood cell (RBC) membrane and plasma. Precursor FAs dietary intake was measured through food frequency questionnaire and environmental tobacco smoke (ETS) through questionnaires. Linear mixed models were used to test oxylipins with predictors. Results Age associated with 15 n6- and 6 n3-oxylipins; race/ethnicity associated with 3 n6- and 1 n3-oxylipins; sex associated with 2 n6-oxylipins. ETS associated with lipoxin-A4. Oxylipins associated with precursor FAs in plasma more often than RBC. RBC levels and dietary intake of precursor FAs more consistently associated with n3-oxylipins than with n6-oxylipins. Conclusions In healthy children, oxylipin levels change with age. Oxylipins associated with precursor FAs more often in plasma than RBC or diet, suggesting that inflammatory regulation leading to FA release into plasma may also be a determinant of oxylipin generation. Impact This is the first study to examine predictors of oxylipins in healthy children at risk of type 1 diabetes.In healthy children at risk of type 1 diabetes, many oxylipins change with age, and most oxylipins do not differ by sex or race/ethnicity.Environmental tobacco smoke exposure was associated with the presence of lipoxin A4.Omega-6- and omega-3-related oxylipin levels were consistently associated with their respective precursor fatty acid levels measured in the plasma.Proportionally more omega-3 compared to omega-6 oxylipins were associated with dietary intake and red blood cell membrane levels of the respective precursor fatty acid.

We assessed associations between infant diet (e.g., breastfeeding and introduction to solid foods) and DNA methylation in infancy and childhood. We measured DNA methylation in peripheral blood collected in infancy (9–15 months of age) in 243 children, and in a subset of 50 children, we also measured methylation in childhood (6–9 years of age) to examine persistence, and at birth (in cord blood) to examine temporality. We performed multivariable linear regression of infant diet on the outcome of methylation using epigenome-wide and candidate site approaches. We identified six novel CpG sites associated with breastfeeding duration using an EWAS approach. One differentially methylated site presented directionally consistent associations with breastfeeding (cg00574958, CPT1A) in infancy and childhood but not at birth. Two differentially methylated sites in infancy (cg19693031, TXNIP, cg23307264, KHSRP) were associated with breastfeeding and were not present at birth, however, these associations did not persist into childhood. Associations between infant diet and methylation in infancy at three sites (cg22369607, AP001525.1, cg2409200, TBCD, cg27173510, PGBD5) were also present at birth, suggesting the influence of exposures other than infant diet. Infant diet exposures are associated with persistent methylation differences in CPT1A, which may be one mechanism behind infant diet’s long-term health effects.

AIMS/HYPOTHESIS: Oxylipins are lipid mediators derived from polyunsaturated fatty acids. Some oxylipins are proinflammatory (e.g. those derived from arachidonic acid [ARA]), others are pro-resolving of inflammation (e.g. those derived from α-linolenic acid [ALA], docosahexaenoic acid [DHA] and eicosapentaenoic acid [EPA]) and others may be both (e.g. those derived from linoleic acid [LA]). The goal of this study was to examine whether oxylipins are associated with incident type 1 diabetes. METHODS: We conducted a nested case–control analysis in the Diabetes Autoimmunity Study in the Young (DAISY), a prospective cohort study of children at risk of type 1 diabetes. Plasma levels of 14 ARA-derived oxylipins, ten LA-derived oxylipins, six ALA-derived oxylipins, four DHA-derived oxylipins and two EPA-related oxylipins were measured by ultra-HPLC–MS/MS at multiple timepoints related to autoantibody seroconversion in 72 type 1 diabetes cases and 71 control participants, which were frequency matched on age at autoantibody seroconversion (of the case), ethnicity and sample availability. Linear mixed models were used to obtain an age-adjusted mean of each oxylipin prior to type 1 diabetes. Age-adjusted mean oxylipins were tested for association with type 1 diabetes using logistic regression, adjusting for the high risk HLA genotype HLA-DR3/4,DQB1*0302. We also performed principal component analysis of the oxylipins and tested principal components (PCs) for association with type 1 diabetes. Finally, to investigate potential critical timepoints, we examined the association of oxylipins measured before and after autoantibody seroconversion (of the cases) using PCs of the oxylipins at those visits. RESULTS: The ARA-related oxylipin 5-HETE was associated with increased type 1 diabetes risk. Five LA-related oxylipins, two ALA-related oxylipins and one DHA-related oxylipin were associated with decreased type 1 diabetes risk. A profile of elevated LA- and ALA-related oxylipins (PC1) was associated with decreased type 1 diabetes risk (OR 0.61; 95% CI 0.40, 0.94). A profile of elevated ARA-related oxylipins (PC2) was associated with increased diabetes risk (OR 1.53; 95% CI 1.03, 2.29). A critical timepoint analysis showed type 1 diabetes was associated with a high ARA-related oxylipin profile at post-autoantibody-seroconversion but not pre-seroconversion. CONCLUSIONS/INTERPRETATION: The protective association of higher LA- and ALA-related oxylipins demonstrates the importance of both inflammation promotion and resolution in type 1 diabetes. Proinflammatory ARA-related oxylipins may play an important role once the autoimmune process has begun.

Epigenetic mechanisms may contribute to idiopathic scoliosis (IS). We identified 8 monozygotic twin pairs with IS, 6 discordant (Cobb angle difference > 10°) and 2 concordant (Cobb angle difference ≤ 2°). Genome-wide methylation in blood was measured with the Infinium HumanMethylation EPIC Beadchip. We tested for differences in methylation and methylation variability between discordant twins and tested the association between methylation and curve severity in all twins. Differentially methylated region (DMR) analyses identified gene promoter regions. Methylation at cg12959265 (chr. 7 DPY19L1) was less variable in cases (false discovery rate (FDR) = 0.0791). We identified four probes (false discovery rate, FDR < 0.10); cg02477677 (chr. 17, RARA gene), cg12922161 (chr. 2 LOC150622 gene), cg08826461 (chr. 2), and cg16382077 (chr. 7) associated with curve severity. We identified 57 DMRs where hyper- or hypo-methylation was consistent across the region and 28 DMRs with a consistent association with curve severity. Among DMRs, 21 were correlated with bone methylation. Prioritization of regions based on methylation concordance in bone identified promoter regions for WNT10A (WNT signaling), NPY (regulator of bone and energy homeostasis), and others predicted to be relevant for bone formation/remodeling. These regions may aid in understanding the complex interplay between genetics, environment, and IS.

Environmental factors including viruses, diet, and the metabolome have been linked with the appearance of islet autoimmunity (IA) that precedes development of type 1 diabetes (T1D). We measured global DNA methylation (DNAm) and untargeted metabolomics prior to IA and at the time of seroconversion to IA in 92 IA cases and 91 controls from the Diabetes Autoimmunity Study in the Young (DAISY). Causal mediation models were used to identify seven DNAm probe-metabolite pairs in which the metabolite measured at IA mediated the protective effect of the DNAm probe measured prior to IA against IA risk. These pairs included five DNAm probes mediated by histidine (a metabolite known to affect T1D risk), one probe (cg01604946) mediated by phostidyl choline p-32:0 or o-32:1, and one probe (cg00390143) mediated by sphingomyelin d34:2. The top 100 DNAm probes were over-represented in six reactome pathways at the FDR &lt, 0.1 level (q = 0.071), including transport of small molecules and inositol phosphate metabolism. While the causal pathways in our mediation models require further investigation to better understand the biological mechanisms, we identified seven methylation sites that may improve our understanding of epigenetic protection against T1D as mediated by the metabolome.

Background: Diastolic dysfunction (DD) is associated with the development of heart failure and contributes to the pathogenesis of other cardiac maladies, including atrial fibrillation. Inhibition of histone deacetylases (HDACs) has been shown to prevent DD by enhancing myofibril relaxation. We addressed the therapeutic potential of HDAC inhibition in a model of established DD with preserved ejection fraction. Methods: Four weeks after uninephrectomy and implantation with deoxycorticosterone acetate pellets, when DD was clearly evident, 1 cohort of mice was administered the clinical-stage HDAC inhibitor ITF2357/Givinostat. Echocardiography, blood pressure measurements, and end point invasive hemodynamic analyses were performed. Myofibril mechanics and intact cardiomyocyte relaxation were assessed ex vivo. Cardiac fibrosis was evaluated by picrosirius red staining and second harmonic generation microscopy of left ventricle (LV) sections, RNA sequencing of LV mRNA, mass spectrometry–based evaluation of decellularized LV biopsies, and atomic force microscopy determination of LV stiffness. Mechanistic studies were performed with primary rat and human cardiac fibroblasts. Results: HDAC inhibition normalized DD without lowering blood pressure in this model of systemic hypertension. In contrast to previous models, myofibril relaxation was unimpaired in uninephrectomy/deoxycorticosterone acetate mice. Furthermore, cardiac fibrosis was not evident in any mouse cohort on the basis of picrosirius red staining or second harmonic generation microscopy. However, mass spectrometry revealed induction in the expression of >100 extracellular matrix proteins in LVs of uninephrectomy/deoxycorticosterone acetate mice, which correlated with profound tissue stiffening based on atomic force microscopy. ITF2357/Givinostat treatment blocked extracellular matrix expansion and LV stiffening. The HDAC inhibitor was subsequently shown to suppress cardiac fibroblast activation, at least in part, by blunting recruitment of the profibrotic chromatin reader protein BRD4 (bromodomain-containing protein 4) to key gene regulatory elements. Conclusions: These findings demonstrate the potential of HDAC inhibition as a therapeutic intervention to reverse existing DD and establish blockade of extracellular matrix remodeling as a second mechanism by which HDAC inhibitors improve ventricular filling. Our data reveal the existence of pathophysiologically relevant covert or hidden cardiac fibrosis that is below the limit of detection of histochemical stains such as picrosirius red, highlighting the need to evaluate fibrosis of the heart using diverse methodologies.

Rationale: Allergic diseases are an increasing public health concern, and early life environment is critical to immune development. Maternal diet during pregnancy has been linked to offspring allergy risk. In turn, maternal diet is a potentially modifiable factor, which could be targeted as an allergy prevention strategy. In this systematic review, we focused on non-allergen-specific modifying factors of the maternal diet in pregnancy on allergy outcomes in their offspring. Methods: We undertook a systematic review of studies investigating the association between maternal diet during pregnancy and allergic outcomes (asthma/wheeze, hay fever/allergic rhinitis/seasonal allergies, eczema/atopic dermatitis (AD), food allergies, and allergic sensitization) in offspring. Studies evaluating the effect of food allergen intake were excluded. We searched three bibliographic databases (MEDLINE, EMBASE, and Web of Science) through February 26, 2019. Evidence was critically appraised using modified versions of the Cochrane Collaboration Risk of Bias tool for intervention trials and the National Institute for Clinical Excellence methodological checklist for cohort and case-control studies and meta-analysis performed from RCTs. Results: We identified 95 papers: 17 RCTs and 78 observational (case-control, cross-sectional, and cohort) studies. Observational studies varied in design and dietary intakes and often had contradictory findings. Based on our meta-analysis, RCTs showed that vitamin D supplementation (OR: 0.72; 95% CI: 0.56-0.92) is associated with a reduced risk of wheeze/asthma. A positive trend for omega-3 fatty acids was observed for asthma/wheeze, but this did not reach statistical significance (OR: 0.70; 95% CI: 0.45-1.08). Omega-3 supplementation was also associated with a non-significant decreased risk of allergic rhinitis (OR: 0.76; 95% CI: 0.56-1.04). Neither vitamin D nor omega-3 fatty acids were associated with an altered risk of AD or food allergy. Conclusions: Prenatal supplementation with vitamin D may have beneficial effects for prevention of asthma. Additional nutritional factors seem to be required for modulating the risk of skin and gastrointestinal outcomes. We found no consistent evidence regarding other dietary factors, perhaps due to differences in study design and host features that were not considered. While confirmatory studies are required, there is also a need for performing RCTs beyond single nutrients/foods.

Phthalates are known endocrine disruptors and associated with decreased fecundity, pregnancy loss, and adverse obstetrical outcomes, however the underlying mechanisms remain to be established. Environmental factors can influence gene expression and cell function by modifying epigenetic marks, impacting the developing embryo as well as future generations of offspring. The impact of phthalates on placental gene methylation and expression is largely unknown. We studied the effect of maternal phthalate exposure on the human placental DNA methylome and transcriptome. We determined epigenome-wide DNA methylation marks (Illumina Infinium Human Methylation 850k BeadChip) and gene expression (Agilent whole human genome array) associated with phthalate exposure in first trimester placenta. Integrative genomic analysis of candidate genes was performed to define gene methylation-expression relationships. We identified 39 genes with significantly altered methylation and gene expression in the high phthalate exposure group. Most of these relationships were inversely correlated. This analysis identified epidermal growth factor receptor (EGFR) as a critical candidate gene mediating the effects of phthalates on early placental function. Although additional studies are needed to determine the functional consequences of these changes, our findings are consistent with the model that phthalates impact placental function by modulating the expression of critical placental genes through epigenetic regulation.

The intrauterine period is a critical time wherein developmental exposure can influence risk for chronic disease including childhood obesity. Using umbilical cord-derived mesenchymal stem cells (uMSC) from offspring born to normal-weight and obese mothers, we tested the hypothesis that changes in infant body composition over the first 5 months of life correspond with differences in cellular metabolism and transcriptomic profiles at birth. Higher long-chain acylcarnitine concentrations, lipid transport gene expression, and indicators of oxidative stress in uMSC-adipocytes were related to higher adiposity at 5 months of age. In uMSC-myocytes, lower amino acid concentrations and global differential gene expression for myocyte growth, amino acid biosynthesis, and oxidative stress were related to lower infant percent fat-free mass at 5 months of age, particularly in offspring of obese mothers. This is the first evidence of human infant adipocyte- or myocyte-related alterations in cellular metabolic pathways that correspond with increased adiposity and lower fat-free mass in early infancy. These pathways might reflect the effects of an adverse maternal metabolic environment on the fetal metabolome and genome. Our findings suggest that programmed differences in infant stem cell metabolism correspond with differences in body composition in early life, a known contributor to obesity risk.

Ethyl alcohol is a toxin that, when consumed at high levels, produces organ damage and death. One way to prevent or ameliorate this damage in humans is to reduce the exposure of organs to alcohol by reducing alcohol ingestion. Both the propensity to consume large volumes of alcohol and the susceptibility of human organs to alcohol-induced damage exhibit a strong genetic influence. We have developed an integrative genetic/genomic approach to identify transcriptional networks that predispose complex traits, including propensity for alcohol consumption and propensity for alcohol-induced organ damage. In our approach, the phenotype is assessed in a panel of recombinant inbred (RI) rat strains, and quantitative trait locus (QTL) analysis is performed. Transcriptome data from tissues/organs of naïve RI rat strains are used to identify transcriptional networks using Weighted Gene Coexpression Network Analysis (WGCNA). Correlation of the first principal component of transcriptional coexpression modules with the phenotype across the rat strains, and overlap of QTLs for the phenotype and the QTLs for the coexpression modules (module eigengene QTL) provide the criteria for identification of the functionally related groups of genes that contribute to the phenotype (candidate modules). While we previously identified a brain transcriptional module whose QTL overlapped with a QTL for levels of alcohol consumption in HXB/BXH RI rat strains and 12 selected rat lines, this module did not account for all of the genetic variation in alcohol consumption. Our search for QTL overlap and correlation of coexpression modules with phenotype can, however, be applied to any organ in which the transcriptome has been measured, and this represents a holistic approach in the search for genetic contributors to complex traits. Previous work has implicated liver/brain interactions, particularly involving inflammatory/immune processes, as influencing alcohol consumption levels. We have now analyzed the liver transcriptome of the HXB/BXH RI rat panel in relation to the behavioral trait of alcohol consumption. We used RNA-Seq and microarray data to construct liver transcriptional networks, and identified a liver candidate transcriptional coexpression module that explained 24% of the genetic variance in voluntary alcohol consumption. The transcripts in this module focus attention on liver secretory products that influence inflammatory and immune signaling pathways. We propose that these liver secretory products can interact with brain mechanisms that affect alcohol consumption, and targeting these pathways provides a potential approach to reducing high levels of alcohol intake and also protecting the integrity of the liver and other organs.

DNA methylation may be involved in development of type 1 diabetes (T1D), but previous epigenome-wide association studies were conducted among cases with clinically diagnosed diabetes. Using multiple pre-disease peripheral blood samples on the Illumina 450 K and EPIC platforms, we investigated longitudinal methylation differences between 87 T1D cases and 87 controls from the prospective Diabetes Autoimmunity Study in the Young (DAISY) cohort. Change in methylation with age differed between cases and controls in 10 regions. Average longitudinal methylation differed between cases and controls at two genomic positions and 28 regions. Some methylation differences were detectable and consistent as early as birth, including before and after the onset of preclinical islet autoimmunity. Results map to transcription factors, other protein coding genes, and non-coding regions of the genome with regulatory potential. The identification of methylation differences that predate islet autoimmunity and clinical diagnosis may suggest a role for epigenetics in T1D pathogenesis; however, functional validation is warranted.

Seizures that occur during early development are associated with adverse neurodevelopmental outcomes. Causation and mechanisms are currently under investigation. Induction of an early life seizure by kainic acid (KA) in immature rats on post-natal day (P) 7 results in behavioral changes in the adult rat that reflect social and intellectual deficits without overt cellular damage. Our previous work also demonstrated increased expression of CA1 hippocampal long-term potentiation (LTP) and reduced desensitization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type ionotropic glutamate receptors (AMPA-R) one week following a kainic acid induced seizure (KA-ELS). Here we used RNA sequencing (RNAseq) of mRNA from dorsal hippocampal CA1 to probe changes in mRNA levels one week following KA-ELS as a means to investigate the mechanisms for these functional changes. Ingenuity pathway analysis (IPA) confirmed our previous results by predicting an up-regulation of the synaptic LTP pathway. Differential gene expression results revealed significant differences in 7 gene isoforms. Additional assessments included AMPA-R splice variants and adenosine deaminase acting on RNA 2 (ADAR2) editing sites as a means to determine the mechanism for reduced AMPA-R desensitization. Splice variant analysis demonstrated that KA-ELS result in a small, but significant decrease in the "flop" isoform of Gria3, and editing site analysis revealed significant changes in the editing of a kainate receptor subunit, Grik2, and a serotonin receptor, Htr2c. While these specific changes may not account for altered AMPA-R desensitization, the differences indicate that KA-ELS alters gene expression in the hippocampal CA1 one week after the insult.

BACKGROUND: A statistical pipeline was developed and used for determining candidate genes and candidate gene co-expression networks involved in two alcohol (i.e., ethanol) metabolism phenotypes, namely alcohol clearance and acetate area under the curve (AUC) in a recombinant inbred (HXB/BXH) rat panel. The approach was also used to provide an indication of how ethanol metabolism can impact the normal function of the identified networks. METHODS: RNA was extracted from alcohol-naïve liver tissue of 30 strains of HXB/BXH recombinant inbred rats. The reconstructed transcripts were quantitated and data was used to construct gene co-expression modules and networks. A separate group of rats, comprising the same 30 strains, were injected with ethanol (2 gm/kg) for measurement of blood ethanol and acetate levels. These data were used for QTL analysis of the rate of ethanol disappearance and circulating acetate levels. The analysis pipeline required calculation of the module eigengene values, the correction of these values with ethanol metabolism rates and acetate levels across the rat strains and the determination of the eigengene QTLs. For a module to be considered a candidate for determining phenotype, the module eigengene values had to have significant correlation with the strain phenotypic values and the module eigengene QTLs had to overlap the phenotypic QTLs. RESULTS: Of the 658 transcript co-expression modules generated from liver RNA sequencing data, a single module satisfied all criteria for being a candidate for determining the alcohol clearance trait. This module contained two alcohol dehydrogenase genes, including the gene whose product was previously shown to be responsible for the majority of alcohol elimination in the rat. This module was also the only module identified as a candidate for influencing circulating acetate levels. This module was also linked to the process of generation and utilization of retinoic acid as related to the autonomous immune response. CONCLUSIONS: We propose that our analytical pipeline can successfully identify genetic regions and transcripts which predispose a particular phenotype and our analysis provides functional context for co-expression module components.

A quantitative genetic approach, which involves correlation of transcriptional networks with the phenotype in a recombinant inbred (RI) population and in selectively bred lines of rats, and determination of coinciding quantitative trait loci for gene expression and the trait of interest, has been applied in the present study. In this analysis, a novel approach was used that combined DNA-Seq data, data from brain exon array analysis of HXB/BXH RI rat strains and six pairs of rat lines selectively bred for high and low alcohol preference, and RNA-Seq data (including rat brain transcriptome reconstruction) to quantify transcript expression levels, generate co-expression modules and identify biological functions that contribute to the predisposition of consuming varying amounts of alcohol. A gene co-expression module was identified in the RI rat strains that contained both annotated and unannotated transcripts expressed in the brain, and was associated with alcohol consumption in the RI panel. This module was found to be enriched with differentially expressed genes from the selected lines of rats. The candidate genes within the module and differentially expressed genes between high and low drinking selected lines were associated with glia (microglia and astrocytes) and could be categorized as being related to immune function, energy metabolism and calcium homeostasis, as well as glial-neuronal communication. The results of the present study show that there are multiple combinations of genetic factors that can produce the same phenotypic outcome. Although no single gene accounts for predisposition to a particular level of alcohol consumption in every animal model, coordinated differential expression of subsets of genes in the identified pathways produce similar phenotypic outcomes.The datasets supporting the results of the present study are available at http://phenogen.ucdenver.edu.

The ILSXISS (LXS) recombinant inbred (RI) panel of mice is a valuable resource for genetic mapping studies of complex traits, due to its genetic diversity and large number of strains. Male and female mice from this panel were used to investigate genetic influences on alcohol consumption in the “drinking in the dark” (DID) model. Male mice (38 strains) and female mice (36 strains) were given access to 20 % ethanol during the early phase of their circadian dark cycle for four consecutive days. The first principal component of alcohol consumption measures on days 2, 3, and 4 was used as a phenotype (DID phenotype) to calculate QTLs, using a SNP marker set for the LXS RI panel. Five QTLs were identified, three of which included a significant genotype by sex interaction, i.e., a significant genotype effect in males and not females. To investigate candidate genes associated with the DID phenotype, data from brain microarray analysis (Affymetrix Mouse Exon 1.0 ST Arrays) of male LXS RI strains were combined with RNA-Seq data (mouse brain transcriptome reconstruction) from the parental ILS and ISS strains in order to identify expressed mouse brain transcripts. Candidate genes were determined based on common eQTL and DID phenotype QTL regions and correlation of transcript expression levels with the DID phenotype. The resulting candidate genes (in particular, Arntl/Bmal1) focused attention on the influence of circadian regulation on the variation in the DID phenotype in this population of mice.

Elevated tumor expression of the cell surface GPI-linked CD24 protein signals poor patient prognosis in many tumor types. However, some cancer cells selected to be negative for surface CD24 (surCD24−) still retain aggressive phenotypes in vitro and in vivo. Here, we resolve this apparent paradox with the discovery of biologically active, nuclear CD24 (nucCD24) and finding that its levels are unchanged in surCD24− cells. Using the complementary techniques of biochemical cellular fractionation and immunofluorescence, we demonstrate a signal for CD24 in the nucleus in cells from various histologic types of cancer. Nuclear-specific expression of CD24 (NLS-CD24) increased anchorage-independent growth in vitro and tumor formation in vivo. Immunohistochemistry of patient tumor samples revealed the presence of nucCD24, whose signal intensity correlated positively with the presence of metastatic disease. Analysis of gene expression between cells expressing CD24 and NLS-CD24 revealed a unique nucCD24 transcriptional signature. The median score derived from this signature was able to stratify overall survival in four patient datasets from bladder cancer and five patient datasets from colorectal cancer. Patients with high scores (more nucCD24-like) had reduced survival. These findings define a novel and functionally important intracellular location of CD24; they explain why surCD24− cells can remain aggressive, and they highlight the need to consider nucCD24 in both fundamental research and therapeutic development. Cancer Res; 77(18); 4858–67. ©2017 AACR.

Aminoquinoline derivatives were evaluated against a panel of receptors/channels/transporters in radioligand binding experiments. One of these derivatives (DCUK-OEt) displayed micromolar affinity for brain γ-aminobutyric acid type A (GABAA) receptors. DCUK-OEt was shown to be a positive allosteric modulator (PAM) of GABA currents with α1β2γ2, α1β3γ2, α5β3γ2 and α1β3δ GABAA receptors, while having no significant PAM effect on αβ receptors or α1β1γ2, α1β2γ1, α4β3γ2 or α4β3δ receptors. DCUK-OEt modulation of α1β2γ2 GABAA receptors was not blocked by flumazenil. The subunit requirements for DCUK-OEt actions distinguished DCUK-OEt from other currently known modulators of GABA function (e.g., anesthetics, neurosteroids or ethanol). Simulated docking of DCUK-OEt at the GABAA receptor suggested that its binding site may be at the α + β- subunit interface. In slices of the central amygdala, DCUK-OEt acted primarily on extrasynaptic GABAA receptors containing the α1 subunit and generated increases in extrasynaptic “tonic” current with no significant effect on phasic responses to GABA. DCUK-OEt is a novel chemical structure acting as a PAM at particular GABAA receptors. Given that neurons in the central amygdala responding to DCUK-OEt were recently identified as relevant for alcohol dependence, DCUK-OEt should be further evaluated for the treatment of alcoholism.

Recent understanding of the systems that mediate complex disease states, has generated a search for molecules that simultaneously modulate more than one component of a pathologic pathway. Chronic pain syndromes are etiologically connected to functional changes (sensitization) in both peripheral sensory neurons and in the central nervous system (CNS). These functional changes involve modifications of a significant number of components of signal generating, signal transducing and signal propagating pathways. Our analysis of disease-related changes which take place in sensory neurons during sensitization led to the design of a molecule that would simultaneously inhibit peripheral NMDA receptors and voltage sensitive sodium channels. In the current report, we detail the selectivity of N,N-(diphenyl)-4-ureido-5,7-dichloro-2-carboxy-quinoline (DCUKA) for action at NMDA receptors composed of different subunit combinations and voltage sensitive sodium channels having different α subunits. We show that DCUKA is restricted to the periphery after oral administration, and that circulating blood levels are compatible with its necessary concentrations for effects at the peripheral cognate receptors/channels that were assayed in vitro. Our results demonstrate that DCUKA, at concentrations circulating in the blood after oral administration, can modulate systems which are upregulated during peripheral sensitization, and are important for generating and conducting pain information to the CNS. Furthermore, we demonstrate that DCUKA ameliorates the hyperalgesia of chronic pain without affecting normal pain responses in neuropathic and inflammation-induced chronic pain models.

Breast cancer patients diagnosed within five years following pregnancy have increased metastasis and decreased survival. A hallmark of postpartum biology that may contribute to this poor prognosis is mammary gland involution, involving massive epithelial cell death and dramatic stromal remodeling. Previous studies show pro-tumorigenic properties of extracellular matrix (ECM) isolated from rodent mammary glands undergoing postpartum involution. More recent work demonstrates systemic ibuprofen treatment during involution decreases its tumor-promotional nature. Utilizing a proteomics approach, we identified relative differences in the composition of mammary ECM isolated from nulliparous rats and those undergoing postpartum involution, with and without ibuprofen treatment. GeLC-MS/MS experiments resulted in 20327 peptide identifications that mapped to 884 proteins with a

Background Two features of alcohol addiction that have been widely studied in animal models are relapse drinking following periods of alcohol abstinence and the escalation of alcohol consumption after chronic continuous or intermittent alcohol exposure. The genetic contribution to these phenotypes has not been systematically investigated. Methods HXB/BXH recombinant inbred (RI) rat strains were given access to alcohol sequentially as follows: alcohol (10%) as the only fluid for 1 week; alcohol (10%) and water in a 2-bottle choice paradigm for 7 weeks (“pre-alcohol deprivation effect [ADE] alcohol consumption”); 2 weeks of access to water only (alcohol deprivation); and 2 weeks of reaccess to 10% alcohol and water (“post-ADE alcohol consumption”). The periods of deprivation and reaccess to alcohol were repeated 3 times. The ADE was defined as the amount of alcohol consumed in the first 24 hours after deprivation minus the average daily amount of alcohol consumed in the week prior to deprivation. Heritability of the phenotypes was determined by analysis of variance, and quantitative trait loci (QTLs) were identified. Results All strains showed increased alcohol consumption, compared to the predeprivation period, in the first 24 hours after each deprivation (ADE). Broad-sense heritability of the ADEs was low (ADE1, 9.1%; ADE2, 26.2%; ADE3, 16.3%). Alcohol consumption levels were relatively stable over weeks 2 to 7. Post-ADE alcohol consumption levels consistently increased in some strains and were decreased or unchanged in others. Heritability of pre- and post-ADE alcohol consumption was high and increased over time (week 2, 38.5%; week 7, 51.1%; week 11, 56.8%; week 15, 63.3%). QTLs for pre- and post-ADE alcohol consumption were similar, but the strength of the QTL association with the phenotype decreased over time. Conclusions In the HXB/BXH RI rat strains, genotypic variance does not account for a large proportion of phenotypic variance in the ADE phenotype (low heritability), suggesting a role of environmental factors. In contrast, a large proportion of the variance across the RI strains in pre- and post-ADE alcohol consumption is due to genetically determined variance (high heritability).