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PDF file - 614K, Supplementary Table S1 provides data on the Esr2 genotype composition, liter size and weight of offspring at weaning and at 10 months. Supplementary Tables S2 and S3 provide statistical data for analyses of survival and lung tumorigenesis. Supplementary Table 4 provides data on liver tumor outcome. Supplementary Figure S1 summarizes the experiment design. Illustrative histopathology images are provided in Supplementary Figure S2. And results of main effects of experimental factors carcinogen, diet, genotype and gender are summarized in Supplementary Figure S3.

Consumption of the total Western diet (TWD) in mice has been shown to increase gut inflammation, promote colon tumorigenesis, and alter fecal microbiome composition when compared to mice fed a healthy diet, i.e., AIN93G (AIN). However, it is unclear whether the gut microbiome contributes directly to colitis-associated CRC in this model. The objective of this study was to determine whether dynamic fecal microbiota transfer (FMT) from donor mice fed either the AIN basal diet or the TWD would alter colitis symptoms or colitis-associated CRC in recipient mice, which were fed either the AIN diet or the TWD, using a 2 × 2 factorial experiment design. Time-matched FMT from the donor mice fed the TWD did not significantly enhance symptoms of colitis, colon epithelial inflammation, mucosal injury, or colon tumor burden in the recipient mice fed the AIN diet. Conversely, FMT from the AIN-fed donors did not impart a protective effect on the recipient mice fed the TWD. Likewise, the composition of fecal microbiomes of the recipient mice was also affected to a much greater extent by the diet they consumed than by the source of FMT. In summary, FMT from the donor mice fed either basal diet with differing colitis or tumor outcomes did not shift colitis symptoms or colon tumorigenesis in the recipient mice, regardless of the basal diet they consumed. These observations suggest that the gut microbiome may not contribute directly to the development of disease in this animal model.

Carbon monoxide (CO) is a gaseous signaling molecule produced in humans via the breakdown of heme in an O(2)-dependent reaction catalyzed by heme oxygenase enzymes. A long-lived species relative to other signaling molecules (e.g., NO, H(2)S), CO exerts its physiological effects via binding to low-valent transition metal centers in proteins and enzymes. Studies involving the administration of low doses of CO have shown its potential as a therapeutic agent to produce vasodilation, anti-inflammatory, anti-apoptotic, and anti-cancer effects. In pursuit of developing tools to define better the role and therapeutic potential of CO, carbon monoxide releasing molecules (CORMs) were developed. To date, the vast majority of reported CORMs have been metal carbonyl complexes, with the most well-known being Ru(2)Cl(4)(CO)(6) (CORM-2), Ru(CO)(3)Cl(glycinate) (CORM-3), and Mn(CO)(4)(S(2)CNMe(CH(2)CO(2)H)) (CORM-401). These complexes have been used to probe the effects of CO in hundreds of cell- and animal-based experiments. However, through recent investigations, it has become evident that these reagents exhibit complicated reactivity in biological environments. The interpretation of the effects produced by some of these complexes is obscured by protein binding, such that their formulation is not clear, and by CO leakage and potential redox activity. An additional weakness with regard to CORM-2 and CORM-3 is that these compounds cannot be tracked via fluorescence. Therefore, it is unclear where or when CO release occurs, which confounds the interpretation of experiments using these molecules. To address these weaknesses, our research team has pioneered the development of metal-free CORMs based on structurally-tunable extended flavonol or quinolone scaffolds. In addition to being highly controlled, with CO release only occurring upon triggering with visible light (photoCORMs), these CO donors are trackable via fluorescence prior to CO release in cellular environments and can be targeted to specific cellular locations. In the Account, we highlight the development and application of a series of structurally-related flavonol photoCORMs that: (1) sense characteristics of cellular environments prior to CO release; (2) enable evaluation of the influence of cytosolic versus mitochondrial-localized CO release on cellular bioenergetics; (3) probe the cytotoxicity and anti-inflammatory effects of intracellular versus extracellular CO delivery; and (4) demonstrate that albumin delivery of a photoCORM enables potent anti-cancer and anti-inflammatory effects. A key advantage of using triggered CO release compounds in these investigations is the ability to examine the effects of the molecular delivery vehicle in the absence and presence of localized CO release, thus providing insight into the independent contributions of CO. Overall, flavonol-based CO delivery molecules offer opportunities for triggerable, trackable, and targetable CO delivery that are unprecedented in terms of previously reported CORMs and, thus, offer significant potential for applications in biological systems.

Black raspberries (BRB) are rich in anthocyanins with purported anti-inflammatory properties. However, it is not known whether dietary supplementation would ameliorate Western-diet enhanced gut inflammation and colon tumorigenesis. We employed a mouse model of colitis-associated colorectal cancer (CAC) to determine the effects of dietary supplementation with 5 to 10% (w/w) whole, freeze-dried BRB in male C57BL/6J mice fed either a standard healthy diet (AIN93G) or the total Western diet (TWD). In a pilot study, BRB suppressed colitis and colon tumorigenesis while also shifting the composition of the fecal microbiome in favor of taxa with purported health benefits, including Bifidobacterium pseudolongum. In a follow-up experiment using a 2 × 2 factorial design with AIN and TWD basal diets with and without 10% (w/w) BRB, supplementation with BRB reduced tumor multiplicity and increased colon length, irrespective of the basal diet, but it did not apparently affect colitis symptoms, colon inflammation or mucosal injury based on histopathological findings. However, BRB intake increased alpha diversity, altered beta diversity and changed the relative abundance of Erysipelotrichaceae, Bifidobacteriaceae, Streptococcaceae, Rikenellaceae, Ruminococcaceae and Akkermansiaceae, among others, of the fecal microbiome. Notably, changes in microbiome profiles were inconsistent with respect to the basal diet consumed. Overall, these studies provide equivocal evidence for in vivo anti-inflammatory effects of BRB on colitis and colon tumorigenesis; yet, BRB supplementation led to dynamic changes in the fecal microbiome composition over the course of disease development.

OBJECTIVES: Anti-inflammatory bioactives in black raspberries (BRB) have been shown to have protective effects on the colon epithelium and may influence gut microbiome. The goal of this study was to determine the effects of dietary intervention with BRB on the dynamic composition of the gut microbiome composition in mice. METHODS: Using a 2 × 2 factorial design, C57BL/6J male mice were fed the standard AIN93G diet or the total Western diet (TWD) for 16 weeks with or without 10% (w/w) whole, freeze-dried BRB powder. The azoxymethane + dextran sodium sulfate model of inflammation-associated colorectal cancer was employed to assess the dynamic response of the gut microbiome to basal diet and BRB treatment prior to, during, and after active colitis and at the study end. Microbiome composition was determined using 16s rRNA sequencing followed by diversity analyses (alpha and beta) and identification of discriminating taxa by with linear discriminant analyses by effect size (lefse). RESULTS: Alpha diversity was markedly reduced during colitis for mice consuming either AIN93G or TWD, with some improvement noted by the recovery phase. Of note, consumption of BRB for two weeks significantly increased alpha diversity measures, and BRB improved alpha diversity in mice fed the AIN93G diet during colitis. Alternatively, BRB appeared less effective in mice fed TWD. Beta diversity was also significantly affected with notable clustering of microbiomes by BRB treatment during and after colitis. Consumption of BRB affected the relative abundance of several key taxa over the course of colitis and recovery from gut injury, including Erysipelotrichaceae, Bifidobacteriaceae, Streptococcaceae, Rikenellaceae, Ruminococcaceae and Akkermansiaceae, among others. CONCLUSIONS: Dietary supplementation with BRB shifted the composition of the gut microbiome during colitis and recovery from gut injury, though the effects were inconsistent with respect to the basal diet consumed. FUNDING SOURCES: USDA NIFA AFRI grant no. 2018-67017-27,516 and 2014-67017-21755.

Production of embryos with high developmental competence by somatic cell nuclear transfer (scNT) is far less efficient than for in vitro fertilized (IVF) embryos, likely due to an accumulation of errors in genome reprogramming that results in aberrant expression of RNA transcripts, including messenger RNAs (mRNA) and, possibly, microRNAs (miRNA). Thus, our objectives were to use RNAseq to determine the dynamics of mRNA expression in early developing scNT and IVF embryos in the context of the maternal-to-embryonic transition (MET) and to correlate apparent transcriptional dysregulation in cloned embryos with miRNA expression profiles. Comparisons between scNT and IVF embryos indicated large scale transcriptome differences, which were most evident at the 8-cell and morula stages for genes associated with biological functions critical for the MET. For two miRNAs previously identified as differentially expressed in scNT morulae, miR-34a and miR-345, negative correlations with some predicted mRNA targets were apparent, though not widespread among the majority of predicted targets. Moreover, although large-scale aberrations in expression of mRNAs were evident during the MET in cattle scNT embryos, these changes were not consistently correlated with aberrations in miRNA expression at the same developmental stage, suggesting that other mechanisms controlling gene expression may be involved.

The efficiency of somatic cell nuclear transfer (scNT) for production of viable offspring is relatively low as compared to in vitro fertilization (IVF), presumably due to deficiencies in epigenetic reprogramming of the donor cell genome. Such defects may also involve the population of small non-coding RNAs (sncRNAs), which are important during early embryonic development. The objective of this study was to examine dynamic changes in relative abundance of sncRNAs during the maternal-to-embryonic transition (MET) in bovine embryos produced by scNT as compared to IVF by using RNA sequencing. When comparing populations of miRNA in scNT versus IVF embryos, only miR-2340, miR-345, and miR34a were differentially expressed in morulae, though many more miRNAs were differentially expressed when comparing across developmental stages. Also of interest, distinct populations of piwi-interacting like RNAs (pilRNAs) were identified in bovine embryos prior to and during embryonic genome activation (EGA) as compared bovine embryos post-EGA and differentiated cells. Overall, sncRNA sequencing analysis of preimplantation embryos revealed largely similar profiles of sncRNAs for IVF and scNT embryos at the 2-cell, 8-cell, morula, and blastocyst stages of development. However, these sncRNA profiles, including miRNA, piRNA, and tRNA fragments, were notably distinct prior to and after completion of the MET. Summary sentence Small non-coding RNA sequencing analysis revealed largely similar sncRNA for IVF and scNT embryos at the 2-cell, 8-cell, morula and blastocyst stages of development. Graphical Abstract

Inhalation of crystalline silica (cSiO(2)) in the workplace is etiologically linked to lupus and other autoimmune diseases. Exposing lupus-prone NZBWF1 mice to respirable cSiO(2) unleashes a vicious cycle of inflammation and cell death in the lung that triggers interferon-regulated gene expression, ectopic lymphoid structure (ELS) development, elevation of local and systemic autoantibodies (AAbs), and glomerulonephritis. However, cSiO(2)-induced inflammation and onset of autoimmunity can be prevented by inclusion of the ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) into diet of these mice. Since cSiO(2) both causes cell death and interferes with efferocytosis, secondary necrosis of residual cell corpses might provide a rich and varied autoantigen (AAg) source in the lung. While it is known that the particle induces anti-nuclear and anti-dsDNA AAbs in NZBWF1 mice, the full extent of the cSiO(2)-induced AAb response relative to specificity and isotype is not yet understood. The purpose of this study was to test the hypotheses that cSiO(2) exposure induces a wide spectrum of AAbs in the pulmonary and systemic compartments, and that dietary DHA intervention prevents these changes. Archived tissue fluid samples were obtained from a prior study in which NZBWF1 mice were fed purified isocaloric diets containing no DHA (control) or DHA corresponding calorically to human doses of 2 and 5 g/day. Mice were intranasally instilled with 1 mg cSiO(2) or saline vehicle weekly for 4 weeks, then groups euthanized 1, 5, 9, or 13 weeks post-instillation (PI) of the last cSiO(2) dose. Bronchoalveolar lavage fluid (BALF) and plasma from each time point were subjected to AAb profiling using a microarray containing 122 AAgs. cSiO(2) triggered robust IgG and IgM AAb responses against lupus-associated AAgs, including DNA, histones, ribonucleoprotein, Smith antigen, Ro/SSA, La/SSB, and complement as early as 1 week PI in BALF and 5 weeks PI in plasma, peaking at 9 and 13 weeks PI, respectively. Importantly, cSiO(2) also induced AAbs to AAgs associated with rheumatoid arthritis (collagen II, fibrinogen IV, fibrinogen S, fibronectin, and vimentin), Sjögren's syndrome (α-fodrin), systemic sclerosis (topoisomerase I), vasculitis (MPO and PR3), myositis (Mi-2, TIF1-γ, MDA5), autoimmune hepatitis (LC-1), and celiac disease (TTG). cSiO(2) elicited comparable but more modest IgA AAb responses in BALF and plasma. cSiO(2)-induced AAb production was strongly associated with time-dependent inflammatory/autoimmune gene expression, ELS development, and glomerulonephritis. AAb responses were dose-dependently suppressed by DHA supplementation and negatively correlated with the ω-3 index, an erythrocyte biomarker of ω-3 content in tissue phospholipids. Taken together, these findings suggest that cSiO(2) exposure elicits a diverse multi-isotype repertoire of AAbs, many of which have been reported in individuals with lupus and other autoimmune diseases. Furthermore, induction of this broad AAb spectrum could be impeded by increasing ω-3 tissue content via dietary DHA supplementation.

OBJECTIVES: Consumption of a Western type diet is a known risk factor for colorectal cancer. Our group previously developed the total Western diet (TWD) for rodents with energy and nutrient profiles that emulate a typical Western diet. In prior studies, consumption of the TWD in C57BL6/J mice markedly enhanced gut inflammation and colon tumorigenesis. In this study, our objective was to assess the dynamics of immune and cancer pathway gene expression during the onset and resolution of DSS-induced colitis. METHODS: Male C57BL/6 J mice were fed the standard AIN93G diet or the TWD diet for 45 days; on day 21, mice were provided 1% (w/V) DSS in the drinking water for 10 days. On days 33 and 45, the colitis disease activity index was determined. Also, days 21, 33, and 45, colon mucosa was collected (n = 8 per group) and total mRNA was isolated for gene expression analysis using the NanoString nCounter Mouse PanCancer Immune Profiling Panel, which targets 750 cancer- and immune-related genes. RESULTS: Results of these targeted gene expression analyses point to striking up-regulation of hundreds of genes associated with interferon response, inflammation, innate immunity, adaptive immunity, and chemokines and receptor pathways in mice fed TWD as compared to the standard AIN diet during active colitis. In a pattern that mirrored the persistent elevation in inflammation and mucosal injury observed in our prior longitudinal studies with TWD and DSS exposure, dysregulation of many of these genes persisted through recovery from gut injury, in addition to the stimulation of other pathways, such as B-cell activation and antigen processing. CONCLUSIONS: This study is the first to assess the dynamics of immune and cancer pathway gene expression during the onset and resolution of DSS-induced colitis and the first to employ highly multiplexed, direct digital detection NanoString technology for analysis of a colitis transcriptome in mouse colon mucosa. Our observations indicate that consumption of the TWD markedly enhanced colitis, delayed recovery from gut injury, and enhanced colon tumorigenesis likely via extensive changes in expression of immune-related genes in the colon mucosa. FUNDING SOURCES: Supported by the Hatch Capacity Project from the USDA National Institute of Food and Agriculture and the Dr. Robert and Carol Deibel Family Endowment.

Lupus is a debilitating multi-organ autoimmune disease clinically typified by periods of flare and remission. Exposing lupus-prone female NZBWF1 mice to crystalline silica (cSiO2), a known human autoimmune trigger, mimics flaring by inducing interferon-related gene (IRG) expression, inflammation, ectopic lymphoid structure (ELS) development, and autoantibody production in the lung that collectively accelerate glomerulonephritis. cSiO2-triggered flaring in this model can be prevented by supplementing mouse diet with the ω-3 polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA). A limitation of previous studies was the use of purified diet that, although optimized for rodent health, does not reflect the high American intake of saturated fatty acid (SFA), ω-6 PUFAs, and total fat. To address this, we employed here a modified Total Western Diet (mTWD) emulating the 50th percentile U.S. macronutrient distribution to discern how DHA supplementation and/or SFA and ω-6 reduction influences cSiO2-triggered lupus flaring in female NZBWF1 mice. Six-week-old mice were fed isocaloric experimental diets for 2 wks, intranasally instilled with cSiO2 or saline vehicle weekly for 4 wks, and tissues assessed for lupus endpoints 11 wks following cSiO2 instillation. In mice fed basal mTWD, cSiO2 induced robust IRG expression, proinflammatory cytokine and chemokine elevation, leukocyte infiltration, ELS neogenesis, and autoantibody production in the lung, as well as early kidney nephritis onset compared to vehicle-treated mice fed mTWD. Consumption of mTWD containing DHA at the caloric equivalent to a human dose of 5 g/day dramatically suppressed induction of all lupus-associated endpoints. While decreasing SFA and ω-6 in mTWD modestly inhibited some disease markers, DHA addition to this diet was required for maximal protection against lupus development. Taken together, DHA supplementation at a translationally relevant dose was highly effective in preventing cSiO2-triggered lupus flaring in NZBWF1 mice, even against the background of a typical Western diet.

Carbon monoxide (CO) is a signaling molecule in humans. Prior research suggests that therapeutic levels of CO can have beneficial effects in treating a variety of physiological and pathological conditions. To facilitate understanding of the role of CO in biology, molecules that enable fluorescence detection of CO in living systems have emerged as an important class of chemical tools. A key unmet challenge in this field is the development of fluorescent analyte replacement probes that replenish the CO that is consumed during detection. Herein, we report the first examples of CO sense and release molecules that involve combining a common CO-sensing motif with a light-triggered CO-releasing flavonol scaffold. A notable advantage of the flavonol-based CO sense and release motif is that it is trackable via fluorescence in both its pre- and postsensing (pre-CO release) forms. In vitro studies revealed that the PdCl2 and Ru(II)-containing CORM-2 used in the CO sensing step can result in metal coordination to the flavonol, which minimizes the subsequent CO release reactivity. However, CO detection followed by CO release is demonstrated in living cells, indicating that a cellular environment mitigates the flavonol/metal interactions.

In mammals, small non-coding RNAs (sncRNAs) have been reported to be important during early embryo development. However, a comprehensive assessment of the inventory of sncRNAs during the maternal-to-zygotic transition (MZT) has not been performed in an animal model that better represents the sncRNA biogenesis pathway in human oocytes and embryos. The objective of this study was to examine dynamic changes in expression of sncRNAs during the MZT in bovine embryos produced by in vitro fertilization (IVF), which occurs at the 8-cell stage. An unbiased, discovery-based approach was employed using small RNAseq to profile sncRNAs in bovine oocytes, 8-cell stage embryos and blastocyst stage embryos followed by network and ontology analyses to explore the functional relevance of differentially expressed micro-RNAS (miRNAs). The relative abundance of miRNAs was markedly higher in 8-cell stage embryos compared to oocytes or blastocyst stage embryos. This shift in miRNA population was largely associated with upregulation of miRNAs predicted to target genes involved in the biological processes of cell development, cell division, Wnt signaling, and pluripotency, among others. Distinct populations of piwi-interacting-like RNAs (pilRNAs) were identified in bovine oocytes and blastocyst stage embryos, though pilRNAs were nearly absent in 8-cell stage embryos. Also, small nucleolar RNAs were highly expressed in 8-cell stage embryos. Overall, these data reveal a strong dynamic shift in the relative abundance of sncRNAs associated with the MZT in bovine oocytes and embryos, suggesting that these molecules may play important roles in the shift from maternal to zygotic control of gene expression.Summary SentenceIn bovine oocytes and IVF embryos, the maternal-to-zygotic transition is associated with a marked shift in abundance of specific sncRNA classes, including miRNAs, pilRNAs, and snoRNAs.

The delivery of controlled amounts of carbon monoxide (CO) to biological targets is of significant current interest. Very few CO-releasing compounds are currently known that can be rigorously controlled in terms of the location and amount of CO released. To address this deficiency, we report herein a new metal-free, visible light-induced CO-releasing molecule (photoCORM) and its prodrug oxidized form, which offer new approaches to controlled, localized CO-delivery. The new photoCORM, based on a 3-hydroxybenzo[g]quinolone framework, releases one equivalent of CO upon visible light illumination under a variety of biologically-relevant conditions. This non-toxic compound can be tracked prior to CO release using fluorescence microscopy and produces a non-toxic byproduct following CO release. An oxidized prodrug form of the photoCORM is reduced by cellular thiols, providing an approach toward activation in the reducing environment of cancer cells. Strong non-covalent affinity of the non-metal photoCORM to albumin enables use of an albumin:photoCORM complex for targeted CO delivery to cancer cells. This approach produced cytotoxicity IC(50) values among the lowest reported to date for CO delivery to cancer cells by a photoCORM. This albumin:photoCORM complex is also the first CO delivery system to produce significant anti-inflammatory effects when introduced at nanomolar photoCORM concentration.

Signaling molecules, including H2S and CO, are involved in a complex interplay within cells to maintain cellular homeostasis. In order to investigate the intracellular interplay of different gasotransmitters, new molecular tools are needed that combine the sensing and release capabilities of different small molecules in a single, multifunctional, and highly regulated molecular platform. This report gives the first example of a combined H2S-sensor/CO-releasing molecule. This flavonol-based molecular tool operates intracellularly via a highly regulated AND logic gated input–output sequence and provides fluorescent feedback for the H2S detection and CO release steps. This linear sequence can be combined with a fluorescent molecular sensor for CO detection via a third distinct emission. Overall, this is the first molecular framework that can combine the sensing of H2S with the controlled release of CO, two gaseous molecules that are known to exhibit reciprocal regulation and have overlapping targets in cellular environments.

Polycyclic aromatic hydrocarbons (PAHs) comprise an important class of environmental pollutants that are known to cause lung cancer in animals and are suspected lung carcinogens in humans. Moreover, evidence from cell-based studies points to PAHs as modulators of the epigenome. The objective of this work was to assess patterns of genome-wide DNA methylation in lung tissues of adult offspring initiated in utero with the transplacental PAH carcinogens dibenzo [def,p]chrysene (DBC) or benzo [a]pyrene (BaP). Genome-wide methylation patterns for normal (not exposed), normal adjacent and lung tumor tissues obtained from adult offspring were determined using methylated DNA immunoprecipitation (MeDIP) with the NimbleGen mouse DNA methylation CpG island array. Lung tumor incidence in 45-week old mice initiated with BaP was 32%, much lower than that of the DBC-exposed offspring at 96%. Also, male offspring appeared more susceptible to BaP as compared to females. Distinct patterns of DNA methylation were associated with non-exposed, normal adjacent and adenocarcinoma lung tissues, as determined by principal components, hierarchical clustering and gene ontology analyses. From these methylation profiles, a set of genes of interest was identified that includes potential important targets for epigenetic modification during the process of lung tumorigenesis in animals exposed to environmental PAHs.

Data in this article depict patterns of methylation in lung tissues obtained from the offspring of B6129SF1/J dams and 129S1/SvImJ sires exposed in utero to benzo[ a ]pyrene (BaP) or dibenzo[ def,p ]chrysene (DBC) as compared to non-exposed offspring. Genome-wide methylation of lung tumors in adult offspring was determined using methylated DNA immunoprecipitation (MeDIP) with the NimbleGen mouse DNA methylation CpG island array. This data article refers to the research article “DNA methylation in lung tissues of mouse offspring exposed in utero to polycyclic aromatic hydrocarbons,” [1] in which comprehensive data interpretation and analysis are provided.

Animal models of chronic disease are continuously being refined and have evolved with the goal of increasing the translation of results to human populations. Examples of this progress include transgenic models and germ-free animals conventionalized with human microbiota. The gut microbiome is involved in the etiology of several chronic diseases. Therefore, consideration of the experimental conditions that may affect the gut microbiome in preclinical disease is very important. Of note, diet plays a large role in shaping the gut microbiome and can be a source of variation between animal models and human populations. Traditionally, nutrition researchers have focused on manipulating the macronutrient profile of experimental diets to model diseases such as metabolic syndrome. However, other dietary components found in human foods, but not in animal diets, can have sizable effects on the composition and metabolic capacity of the gut microbiome and, as a consequence, manifestation of the chronic disease being modeled. The purpose of this review is to describe how food matrix food components, including diverse fiber sources, oxidation products from cooking, and dietary fat emulsifiers, shape the composition of the gut microbiome and influence gut health.

Carbon monoxide (CO) is a gasotransmitter produced in humans. An essential unanswered question in the design of carbon monoxide releasing molecules (CORMs) is whether the delivery molecule should be localized extra- or intracellularly to produce desired biological effects. Herein we show that extracellular CO release is less toxic and is sufficient to produce an anti-inflammatory effect similar to that of intracellular CO release at nanomolar concentrations. This information is valuable for the design of CORMs.

OBJECTIVES: Approximately 1.4 million people suffer from inflammatory bowel disease, which is a major risk factor for developing colitis associated colorectal cancer (CAC). Dietary interventions with the goal to reduce colon inflammation and encourage gut microbiome homeostasis may be a strategy to reduce the risk of CAC. The antioxidants and anti-inflammatory compounds present in black raspberries (BRB) have demonstrated protective effects in the colon epithelium and may alter the composition of the gut microbiome. Previously, we showed that dietary supplementation with black raspberries significantly suppressed colitis and colon tumorigenesis promoted by the consumption of a Western type diet in mice. The goal of this study was to compare the efficacy of dietary intervention with whole, freeze-dried black raspberries on colitis and colon tumorigenesis in mice consuming either a standard diet or a Western type diet that emulates typical U.S. nutrient intakes. METHODS: C57BL/6 J male and female mice were fed a standard diet (AIN93G) or the total Western diet (TWD) supplemented with 0 to 10% (w/w) black raspberry powder for a total of 16 weeks. All mice were dosed with axozymethane and provided 1% dextran sodium sulfate in drinking water for 10 days to promote colonic inflammation and tumorigenesis. RESULTS: As previously observed, mice fed TWD experienced more pronounced symptoms of colitis with a 40% increase in the disease activity index (DAI) score. Preliminary analyses suggest that dietary supplementation with 10% BRB suppressed the DAI score in mice fed TWD such that the colitis symptoms in these mice were not apparently different compared to the AIN93G-fed controls. However, addition of 10% BRB did not appear to provide a benefit to mice fed the AIN basal diet. Composition of the fecal microbiome over the course of disease development will be determined by standard 16S rRNA sequencing, and assessment of tumor outcome is ongoing. CONCLUSIONS: Consumption of a Western type diet increased symptoms of colitis, whereas dietary supplementation with 10% BRB appeared to ameliorate TWD-enhanced colitis in mice. FUNDING SOURCES: USDA NIFA Grant# 2018-67017-27516 and UAES Project UTA-1178.

OBJECTIVES: Dietary strategies to reduce colonic inflammation and promote gut homeostasis may markedly reduce the risk of colitis-associated colorectal cancer. Previously, we showed that dietary supplementation with black raspberries significantly suppressed colitis and colon tumorigenesis promoted by the consumption a Western type diet in mice. In this study, our goal was to assess the impact of consumption of the TWD with and without black raspberry supplementation on the composition of the fecal microbiome over the course of disease development. METHODS: C57BL/6 J male mice were fed a standard diet (AIN93G), the total Western diet (TWD), TWD + 5% (w/w) black raspberry powder (BRB) or TWD + 10% (w/w) BRB for 16 weeks total. After two weeks of feeding test diets, all mice were dosed with axozymethane and provided 1% dextran sodium sulfate in drinking water for 10 days to promote colonic inflammation and tumorigenesis. Composition of the fecal microbiome was determined by standard 16S rRNA sequencing following two weeks of dietary treatment, during active colitis immediately following DSS treatment and again two weeks later during the recovery period. RESULTS: Fecal microbiome profiles in mice fed diets supplemented with 5 or 10% BRB were distinct from those fed AIN93G or TWD diets, shown by significant differences in beta diversity at each of the time points (unweighted unifrac distance, permanova P

Previous work by our group using a mouse model of inflammation-associated colorectal cancer (CAC) showed that the total Western diet (TWD) promoted colon tumor development. Others have also shown that vancomycin-mediated changes to the gut microbiome increased colorectal cancer (CRC). Therefore, the objective of this study was to determine the impact of vancomycin on colon tumorigenesis in the context of a standard mouse diet or the TWD. A 2 × 2 factorial design was used, in which C57Bl/6J mice were fed either the standard AIN93G diet or TWD and with vancomycin in the drinking water or not. While both the TWD and vancomycin treatments independently increased parameters associated with gut inflammation and tumorigenesis compared to AIN93G and plain water controls, mice fed the TWD and treated with vancomycin had significantly increased tumor multiplicity and burden relative to all other treatments. Vancomycin treatment significantly decreased alpha diversity and changed the abundance of several taxa at the phylum, family, and genus levels. Conversely, basal diet had relatively minor effects on the gut microbiome composition. These results support our previous research that the TWD promotes colon tumorigenesis and suggest that vancomycin-induced changes to the gut microbiome are associated with higher tumor rates.

OBJECTIVES: Inhalation of crystalline silica (Si) has been linked to the pathogenesis of human autoimmunity such as lupus. Dietary lipid intervention can delay progression of Si-induced autoimmunity in the lupus-prone female NZBWF1 mice. However, the role of gut microbiome relative to dietary lipids and Si-induced autoimmunity is unknown. We seek to ask whether Si-induced pathogenicity and supplementation of a Western diet with DHA can alter gut microbial populations. METHODS: Four experimental diets were used in this study: modified Total Western Diet (MTWD), MTWD supplemented with DHA (↑DHA), MTWD with reduced saturated and ω-6 fatty acids (↓SFω6), and MTWD with ↓SFω6 and ↑DHA (↓SFω6/↑DHA). Groups of mice (n = 8/gp) were fed one of the four isocaloric experimental diets beginning at 6 wks of age. After 2 wks, mice were anesthetized and intranasally instilled with phosphate-buffered saline (PBS) or Si in PBS. DNA obtained from fecal pellets for gut microbiome analysis was collected from an individual mouse at 13 (early-phase) and 22 (late-phase) wks of age. DNA was extracted and sequenced on the Illumina MiSeq platform. The relative abundance of microbial taxa was differentiated by pairwise comparisons using ANalysis of COmposition of Microbiomes (ANCOM), Linear Mixed model (LM), and LDA Effect Size (LEfSe). RESULTS: Lupus triggering by Si with dietary lipid intervention caused a significant difference in Lachnospiraceae bacterium 609 (Lb609) abundance depending on the stringency of analysis frameworks. Under early-phase data, Lb609 was significantly found to be higher in the group of Si-exposed MTWD (MTWD/Si) than all other groups in LefSe analysis (LDA score >3). LM showed the same results except the comparison between MTWD/Si and ↓SFω6 (FDR-adjust P > 0.05). In the results of ANCOM, Lb609 in MTWD/Si was only significantly different from the groups of PBS-exposed MTWD (MTWD/PBS) and ↓SFω6+↑DHA. Although Lb609 was identified discriminatively between MTWD/Si and MTWD/PBS in all analyses from late-phase data, it was not differential in other comparisons. CONCLUSIONS: Lupus triggering altered microbial composition reflected in increased Lb609 and decreased when supplementation was applied. Further, the studies revealed that interventions on microbial populations were dependent on time. FUNDING SOURCES: The USDA National Institute of Food and Federal Appropriations.

While interactions between carbon monoxide (CO) and mitochondria have been previously studied, the methods used to deliver CO (gas or CO-releasing metal carbonyl compounds) lack subcellular targeting and/or controlled delivery. Thus, the effective concentration needed to produce changes in mitochondrial bioenergetics is yet to be fully defined. To evaluate the influence of mitochondrial-targeted versus intracellularly-released CO on mitochondrial oxygen consumption rates, we developed and characterized flavonol-based CO donor compounds that differ at their site of release. These molecules are metal-free, visible light triggered CO donors (photoCORMs) that quantitatively release CO and are trackable in cells via confocal microscopy. Our studies indicate that at a concentration of 10 μM, the mitochondrial-localized and cytosolic CO-releasing compounds are similarly effective in terms of decreasing ATP production, maximal respiration, and the reserve capacity of A549 cells. This concentration is the lowest to impart changes in mitochondrial bioenergetics for any CO-releasing molecule (CORM) reported to date. The results reported herein demonstrate the feasibility of using a structurally-tunable organic photoCORM framework for comparative intracellular studies of the biological effects of carbon monoxide.

Problem The regulatory mechanisms governing differential expression of classical major histocompatibility complex (MHC) class I (MHC-Ia) and non-classical MHC class I (MHC-Ib) genes are poorly understood. Method of study Quantitative reverse transcription- polymerase chain reaction (PCR) was used to compare the abundance of MHC-I transcripts and related transcription factors in peripheral blood mononuclear cells (PBMC) and placental trophoblast cells (PTC). Methylation of MHC-I CpG islands was detected by bisulfite treatment and next-generation sequencing. Demethylation of PBMC and PTC with 5'-aza-deoxycytidine was used to assess the role of methylation in gene regulation. Results MHC-I expression was higher in PBMC than PTC and was correlated with expression of IRF1, class II MHC transactivator (CIITA), and STAT1. The MHC-Ia genes and BoLA-NC1 were devoid of CpG methylation in PBMC and PTC. In contrast, CpG sites in the gene body of BoLA-NC2, -NC3, and -NC4 were highly methylated in PBMC but largely unmethylated in normal PTC and moderately methylated in somatic cell nuclear transfer PTC. In PBMC, demethylation resulted in upregulation of MHC-Ib by 2.8- to 6-fold, whereas MHC-Ia transcripts were elevated less than 2-fold. Conclusion DNA methylation regulates bovine MHC-Ib expression and is likely responsible for the different relative levels of MHC-Ib to MHC-Ia transcripts in PBMC and PTC.

Rodent models have been invaluable for biomedical research. Preclinical investigations with rodents allow researchers to investigate diseases by using study designs that are not suitable for human subjects. The primary criticism of preclinical animal models is that results are not always translatable to humans. Some of this lack of translation is due to inherent differences between species. However, rodent models have been refined over time, and translatability to humans has improved. Transgenic animals have greatly aided our understanding of interactions between genes and disease and have narrowed the translation gap between humans and model animals. Despite the technological innovations of animal models through advances in genetics, relatively little attention has been given to animal diets. Namely, developing diets that replicate what humans eat will help make animal models more relevant to human populations. This review focuses on commonly used rodent diets that are used to emulate the Western dietary pattern in preclinical studies of obesity and type 2 diabetes, nonalcoholic liver disease, maternal nutrition, and colorectal cancer.

The objectives of this study were to determine the structural characteristics of perfluoroalkyl acids (PFAAs) that confer estrogen-like activity in vivo using juvenile rainbow trout (Oncorhynchus mykiss) as an animal model and to determine whether these chemicals interact directly with the estrogen receptor (ER) using in vitro and in silico species comparison approaches. Perfluorooctanoic (PFOA), perfluorononanoic (PFNA), perfluorodecanoic (PFDA), and perfluoroundecanoic (PFUnDA) acids were all potent inducers of the estrogen-responsive biomarker protein vitellogenin (Vtg) in vivo, although at fairly high dietary exposures. A structure-activity relationship for PFAAs was observed, where eight to ten fluorinated carbons and a carboxylic acid end group were optimal for maximal Vtg induction. These in vivo findings were corroborated by in vitro mechanistic assays for trout and human ER. All PFAAs tested weakly bound to trout liver ER with half maximal inhibitory concentration (IC(50)) values of 15.2-289 μM. Additionally, PFOA, PFNA, PFDA, PFUnDA, and perlfuorooctane sulfonate (PFOS) significantly enhanced human ERα-dependent transcriptional activation at concentrations ranging from 10-1000 nM. Finally, we employed an in silico computational model based upon the crystal structure for the human ERα ligand-binding domain complexed with E2 to structurally investigate binding of these putative ligands to human, mouse, and trout ERα. PFOA, PFNA, PFDA, and PFOS all efficiently docked with ERα from different species and formed a hydrogen bond at residue Arg394/398/407 (human/mouse/trout) in a manner similar to the environmental estrogens bisphenol A and nonylphenol. Overall, these data support the contention that several PFAAs are weak environmental xenoestrogens of potential concern.

Molecular structures capable of intracellular information processing that couple responses from biomarker signals to the release of drug molecules represent intelligent delivery systems. Herein we report a chemophotonically driven, sense-of-logic carbon monoxide-releasing molecule (SL-photoCORM). This extended flavonol motif operates via an AND logic gate by first sensing the cellular environment via detection of thiols and then releasing CO when triggered with visible light and O2. Overall, this approach couples the detection of a cellular redox biomarker with the ability to release a small-molecule gasotransmitter known to trigger pathways involved in pro- and anti-inflammatory effects in a dose-dependent manner. Significantly, the fluorescence properties of the flavonol-based SL-photoCORM produce a series of chemophotonic input responses via two photochromatic switches (blue-to-green and green-to-colorless), leading to trackability and spatiotemporal control of CO release. Examination of the O2 require...

Consumption of a Western type diet is a known risk factor for colorectal cancer. Our group previously developed the total Western diet (TWD) for rodents with energy and nutrient profiles that emulate a typical Western diet. In this study, we tested the hypothesis that consumption of the TWD would enhance colitis, delay recovery from gut injury and promote colon tumorigenesis. In multiple experiments using the azoxymethane + dextran sodium sulfate or ApcMin/+ mouse models of colitis-associated colorectal carcinogenesis (CAC), we determined that mice fed TWD experienced more severe and more prolonged colitis compared to their counterparts fed the standard AIN93G diet, ultimately leading to markedly enhanced colon tumorigenesis. Additionally, this increased tumor response was attributed to the micronutrient fraction of the TWD, and restoration of calcium and vitamin D to standard amounts ameliorated the tumor-promoting effects of TWD. Finally, exposure to the TWD elicited large scale, dynamic changes in mRNA signatures of colon mucosa associated with interferon (IFN) response, inflammation, innate immunity, adaptive immunity, and antigen processing pathways, among others. Taken together, these observations indicate that consumption of the TWD markedly enhanced colitis, delayed recovery from gut injury, and enhanced colon tumorigenesis likely via extensive changes in expression of immune-related genes in the colon mucosa.

Photoactivation is a promising approach for modulating the biological activity of RuII compounds. In this work, RuII flavonolato compounds, [Ru(η6-p-cymene)(L)(3-Hfl)]OTf (8; 3-Hfl = monoanion of 3-hydroxyflavone; L = solvent) and [Ru(η6-p-cymene)Cl(3-Hfl-X)] (3a–3c; 3-Hfl-X = p-H, -Cl, or -F on the flavonolato phenyl substituent), have been evaluated for photoinduced reactivity within the flavonolato unit upon irradiation with UV (300 nm) or visible (419 nm) light under aerobic conditions. For each compound, irradiation in CH3CN was found to result in the loss of the p-cymene ligand and the formation of products resulting from oxidative ring opening of the flavonolato ligand in a dioxygenase-type reaction. This reaction also results in the release of carbon monoxide. The RuII products generated in these reactions are [RuII(solvent)(carboxylato)]+ and [Ru(CO)(solvent)(carboxylato)]+ (carboxylato = O-benzoylsalicylato or benzoato) species, as determined by ESI-MS. The amount of free CO generated depends on...

The Western dietary pattern can alter the gut microbiome and cause obesity and metabolic disorders. To examine the interactions between diet, the microbiome, and obesity, we transplanted gut microbiota from lean or obese human donors into mice fed one of three diets for 22 weeks: (1) a control AIN93G diet, (2) the total Western diet (TWD), which mimics the American diet, or (3) a 45% high-fat diet-induced obesity (DIO) diet. We hypothesized that a fecal microbiome transfer (FMT) from obese donors would lead to an obese phenotype and aberrant glucose metabolism in recipient mice that would be exacerbated by consumption of the TWD or DIO diets. Prior to the FMT, the native microbiome was depleted using an established broad-spectrum antibiotic protocol. Interestingly, the human donor body type microbiome did not significantly affect final body weight or body composition in mice fed any of the experimental diets. Beta diversity analysis and linear discriminant analysis with effect size (LEfSe) showed that mice that received an FMT from obese donors had a significantly different microbiome compared to mice that received an FMT from lean donors. However, after 22 weeks, diet influenced the microbiome composition irrespective of donor body type, suggesting that diet is a key variable in the shaping of the gut microbiome after FMT.

OBJECTIVES: A Western type dietary pattern is a major risk factor for colitis-associated colorectal cancer (CAC). Observations from transgenerational studies suggest that epimutations may be inherited, resulting in persistent aberrant gene expression in offspring. Previously, our group reported that ancestral exposure to the total Western diet (TWD) markedly increased colon cancer incidence and disease severity in F(3) offspring that were not fed this diet directly. Moreover, exposure to TWD over multiple generations markedly exacerbated the disease in F(3) offspring as compared to those fed TWD directly. For the present work, we hypothesized that ancestral or multiple generation exposure to the TWD may result in differential expression of cancer critical genes in such a way that explains the greater tumor abundance and burden observed in these mice. METHODS: C57BL/6 J mice were bred for three generations, during which they were fed a standard diet (AIN93G) for all generations or the total Western diet for rodents (TWD) during only the F(0) generation (ancestral), the F(0) through F(3) generations (multi-generation), or only the F(3) generation (direct). The azoxymethane and dextran sodium sulfate (AOM/DSS) model of CAC was employed in F(3) offspring, from which colon mucosa RNA was isolated and used for Illumina RNAseq with EdgeR for differential expression analysis. RESULTS: About 700 to 4500 differentially expressed genes (DEGs) were identified for colon mucosa from AOM/DSS-initiated offspring compared to their sham controls. For AOM/DSS-initiated mice, >100 DEGs were identified comparing multi-generation TWD-fed mice to their AIN93G-fed counterparts, and 36 genes were different from those direct TWD-fed. Of note, in sham-initiated mice, 101 DEGs were identified comparing direct TWD-fed mice to multi-generation TWD-exposed offspring. Interestingly, these DEGs were associated with defense response, immune response, and response to interferon biological process ontology terms. CONCLUSIONS: Exposure to the TWD over multiple generations caused significant changes in genes involved in immune response in third generation offspring. Assessment of genome-wide patterns of DNA methylation in these colon tissue samples is ongoing. FUNDING SOURCES: USDA NIFA AFRI Grant No. 2014-67017-21755; Utah Agricultural Experiment Station Project UTA-1178.

In the present study, the efficacy of indole-3-carbinol (I3C), a key bioactive component of cruciferous vegetables, for prevention of cancer in offspring exposed in utero to the environmental carcinogen dibenzo[def,p]chrysene (DBC) was evaluated using an estrogen receptor β (ERβ) knockout mouse model. I3C was provided either through the maternal diet coincident with carcinogen exposure during pregnancy or directly to offspring postinitiation with DBC. I3C was effective at reducing T-cell acute lymphoblastic lymphoma/leukemia (T-ALL)–related mortality in offspring only if provided via the maternal diet, although a gender difference in the role of ERβ in mediating this response was evident. In female offspring, chemoprevention of T-ALL by maternal dietary I3C required expression of ERβ; survival in Esr2 wild-type and heterozygous female offspring was more than 90% compared with 66% in Esr2 null females. Alternatively, ERβ status did not significantly impact the transplacental chemoprevention by I3C in males. The possible role of ERβ in mediating lung carcinogenesis or chemoprevention by I3C was similarly complicated. Lung tumor incidence was unaltered by either dietary intervention, whereas lung tumor multiplicity was substantially reduced in Esr2 null females on the control diet and marginally lower in Esr2 null males exposed to I3C via the maternal diet compared with their wild-type and heterozygous counterparts. These findings suggest that I3C may act via ERβ to prevent or suppress DBC-initiated transplacental carcinogenesis but that the involvement of this receptor seems to differ depending on the cancer type and gender of the offspring. Cancer Prev Res; 6(4); 339–48. ©2013 AACR.

cope In pre-clinical studies investigating bioactive components, the efficacy of the bioactive is likely influenced by the basal diet provided to rodents. In this study, we hypothesized that a model bioactive, green tea extract (GTE), would have different effects on colon carcinogenesis, body composition, and lipid metabolism in mice fed a basal diet formulated to promote animal health and growth (AIN93G) as compared to a Western diet that emulates typical American intakes of micro- and macronutrients, the total Western diet (TWD). Methods and results Mice were fed either AIN93G or TWD, with or without GTE added to drinking water for 18 weeks. Aberrant crypt foci (ACF) in azoxymethane-initiated mice was nearly three times greater in mice fed TWD compared to AIN93G. Consumption of GTE suppressed ACF development only in mice fed the TWD. Similarly, supplementation with GTE suppressed weight gain and fasted glucose only in mice fed TWD, while GTE suppressed fat mass in mice fed either diet. Irrespective of diet, GTE supplementation increased cecum weight and decreased cecal SCFA concentration. Conclusion Collectively, these observations indicate that the TWD influences the bioactivity of GTE in rodent models of obesity, metabolism, and carcinogenesis.

In this study, we determined the impact of the total Western diet (TWD) for rodents and its macro- and micronutrient components on weight gain and biomarkers of metabolic function in mice compared to a 45% fat diet-induced obesity (DIO) diet and the standard AIN93G diet. We hypothesized that mice fed the TWD would have increased body fat with indicators of metabolic syndrome similar to mice consuming the DIO diet. As expected, DIO-fed mice acquired a metabolic syndrome phenotype typified by increased energy intake, increased body weight gain, increased fat mass, higher fasting glucose, impaired glucose tolerance, and higher plasma leptin relative to the AIN93G diet. Mice fed a macronutrient-modified (MM) diet (with standard vitamin and mineral composition) had a similar response, albeit to a lesser degree than mice fed the DIO diet. Mice fed a vitamin- and mineral-modified diet (with standard macronutrient composition) were not different from mice fed the AIN93G diet. Surprisingly, the TWD (with modified macronutrients, vitamins and minerals) did not significantly affect any of these parameters, despite the fact that the TWD macronutrient profile was identical to the MM diet. These data suggest that, in the context of the TWD, vitamin and mineral intakes in mice that reflect a Western dietary pattern inhibit the hyperphagia and resulting increased weight gain associated with the higher fat content of the TWD. In conclusion, these observations underscore the need to consider the influence of micronutrient intakes in pre-clinical models of obesity and metabolic syndrome.

Rodent cancer studies typically use defined diets with nutrient profiles optimized for rodent health. However, a defined rodent diet that represents typical American nutrition in all aspects, including calorie sources and macro- and micronutrient composition, is not yet available. Thus, a nutrient density approach was used to formulate the new Total Western Diet (TWD) based on NHANES data for macro- and micronutrient intakes. The TWD has fewer calories from protein and carbohydrate sources and twice that from fat as compared to the AIN-93 diet. The new diet contains more saturated and monounsaturated fats, less polyunsaturated fat, fewer complex carbohydrates, and twice the level of simple sugars. The TWD includes less calcium, copper, folate, thiamin, and vitamins B6, B12, D, and E, but much more sodium. This newly devised diet that better represents typical American nutrition will be highly useful for studies employing animal models of human disease, including cancer.

Previously, we reported that perfluorooctanoic acid (PFOA) promotes liver cancer in a manner similar to that of 17β-estradiol (E2) in rainbow trout. Also, other perfluoroalkyl acids (PFAAs) are weakly estrogenic in trout and bind the trout liver estrogen receptor. The primary objective of this study was to determine whether multiple PFAAs enhance hepatic tumorigenesis in trout, an animal model that represents human insensitivity to peroxisome proliferation. A two-stage chemical carcinogenesis model was employed in trout to evaluate PFOA, perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluorooctane sulfonate (PFOS), and 8:2 fluorotelomer alcohol (8:2FtOH) as complete carcinogens or promoters of aflatoxin B(1) (AFB(1))- and/or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced liver cancer. A custom trout DNA microarray was used to assess hepatic transcriptional response to these dietary treatments in comparison with E2 and the classic peroxisome proliferator, clofibrate (CLOF). Incidence, multiplicity, and size of liver tumors in trout fed diets containing E2, PFOA, PFNA, and PFDA were significantly higher compared with AFB(1)-initiated animals fed control diet, whereas PFOS caused a minor increase in liver tumor incidence. E2 and PFOA also enhanced MNNG-initiated hepatocarcinogenesis. Pearson correlation analyses, unsupervised hierarchical clustering, and principal components analyses showed that the hepatic gene expression profiles for E2 and PFOA, PFNA, PFDA, and PFOS were overall highly similar, though distinct patterns of gene expression were evident for each treatment, particularly for PFNA. Overall, these data suggest that multiple PFAAs can promote liver cancer and that the mechanism of promotion may be similar to that of E2.