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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.

Autoimmune diseases can be triggered by environmental toxicants such as crystalline silica dust (cSiO2). Here, we characterized the dose-dependent immunomodulation and toxicity of the glucocorticoid (GC) prednisone in a preclinical model that emulates onset and progression of cSiO2-triggered lupus. Two cohorts of 6-wk-old female NZBWF1 mice were fed either control AIN-93G diet or one of three AIN-93G diets containing prednisone at 5, 15, or 50 mg/kg diet which span human equivalent oral doses (HED) currently considered to be low (PL; 5 mg/d HED), moderate (PM; 14 mg/d HED), or high (PH; 46 mg/d HED), respectively. At 8 wk of age, mice were intranasally instilled with either saline vehicle or 1 mg cSiO2 once weekly for 4 wk. The experimental plan was to 1) terminate one cohort of mice (n=8/group) 14 wk after the last cSiO2 instillation for pathology and autoimmunity assessment and 2) to maintain a second cohort (n=9/group) to monitor glomerulonephritis development and survival. Mean blood concentrations of prednisone’s principal active metabolite, prednisolone, in mice fed PL, PM, and PH diets were 27, 105, 151 ng/ml, respectively, which are consistent with levels observed in human blood ≤ 12 h after single bolus treatments with equivalent prednisone doses. Results from the first cohort revealed that consumption of PM, but not PL diet, significantly reduced cSiO2-induced pulmonary ectopic lymphoid structure formation, nuclear-specific AAb production, inflammation/autoimmune gene expression in the lung and kidney, splenomegaly, and glomerulonephritis in the kidney. Relative to GC-associated toxicity, PM diet, but not PL diet, elicited muscle wasting, but these diets did not affect bone density or cause glucosuria. Importantly, neither PM nor PL diet improved latency of cSiO2-accelerated death. PH-fed mice in both cohorts displayed robust GC-associated toxicity including body weight loss, reduced muscle mass, and extensive glucosuria 7 wk after the final cSiO2 instillation requiring their early removal from the study. Taken together, our results demonstrate that while moderate doses of prednisone can reduce important pathological endpoints of cSiO2-induced autoimmunity in lupus-prone mice, such as upstream ectopic lymphoid structure formation, these ameliorative effects come with unwanted GC toxicity, and, crucially, none of these three doses extended survival time.

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.

Repeated short-term intranasal instillation of lupus-prone mice with crystalline silica (cSiO2) induces inflammatory gene expression and ectopic lymphoid neogenesis in the lung, leading to early onset of systemic autoimmunity and rapid progression to glomerulonephritis. These responses are suppressed by dietary supplementation with the ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA). Here, we tested the hypothesis that dietary DHA supplementation suppresses cSiO2-induced inflammatory proteins in bronchoalveolar alveolar lavage fluid (BALF) and plasma of lupus-prone mice. Archived tissue fluid samples were used from a prior investigation in which 6 wk-old lupus-prone female NZBWF1 mice were fed isocaloric diets containing 0 or 10 g/kg DHA for 2 wks and then intranasally instilled with 1 mg cSiO2or vehicle once weekly for 4 wks. Cohorts were terminated at 1, 5, 9 or 13 wk post-instillation (PI). BALF and plasma from each cohort were analyzed by high density multiplex array profiling of 200 inflammatory proteins. cSiO2time-dependently induced increases in the BALF protein signatures that were highly reflective of unresolved lung inflammation, although responses in the plasma were much less robust. Induced proteins in BALF included chemokines (e.g., MIP-2, MCP-5), enzymes (e.g., MMP-10, granzyme B), adhesion molecules (e.g., sE-selectin, sVCAM-1), co-stimulatory molecules (e.g., sCD40L, sCD48), TNF superfamily proteins (e.g., sTNFRI, sBAFF-R), growth factors (e.g., IGF-1, IGFBP-3), and signal transduction proteins (e.g., MFG-E8, FcgRIIB), many of which were blocked or delayed by DHA supplementation. The BALF inflammatory proteome correlated positively with prior measurements of gene expression, pulmonary ectopic lymphoid tissue neogenesis, and induction of autoantibodies in the lungs of the control and treatment groups. Ingenuity Pathway Analysis (IPA) revealed that IL-1β, TNF-α, and IL-6 were among the top upstream regulators of the cSiO2-induced protein response. Furthermore, DHA’s effects were associated with downregulation of cSiO2-induced pathways involving i) inhibition of ARE‐mediated mRNA decay, ii) bacterial and viral pattern recognition receptor activation, or iii) TREM1, STAT3, NF-κB, and VEGF signaling and with upregulation of PPAR, LXR/RXR and PPARα/RXRα signaling. Altogether, these preclinical findings further support the contention that dietary DHA supplementation could be applicable as an intervention against inflammation-driven autoimmune triggering by cSiO2or potentially other environmental agents.

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.

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

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.

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.

Crystalline silica (cSiO2) is a widely recognized environmental trigger of autoimmune disease. In the lupus-prone female NZBWF1 mouse, airway exposure to cSiO2 triggers pulmonary ectopic lymphoid neogenesis, systemic autoantibody elevation, and glomerulonephritis. Here we tested the hypothesis that upregulation of adaptive immune function genes in the lung precedes cSiO2-triggering of autoimmune disease in this model. The study include three groups of mice, as follows: (1) necropsied 1 d after a single intranasal instillation of 1 mg cSiO2 or vehicle, (2) necropsied 1 d after four weekly single instillations of 1 mg cSiO2 or vehicle, or (3) necropsied 1, 5, 9, or 13 weeks after four weekly single instillations of 1 mg cSiO2 or vehicle. NanoString nCounter analysis revealed modest transcriptional changes associated with innate and adaptive immune response as early as 1 d after a single cSiO2 instillation. These responses were greatly expanded after four weekly cSiO2 instillations. Concurrent with ectopic lymphoid neogenesis, dramatic increases in mRNAs associated with chemokine release, cytokine production, sustained interferon activity, complement activation, and adhesion molecules were observed. As disease progressed, expression of these genes persisted and was further amplified. Consistent with autoimmune pathogenesis, the time between 5 and 9 weeks post-instillation reflected an important transition period where considerable immune gene upregulation in the lung was observed. Upon termination of the chronic study (13 weeks), cSiO2-induced changes in transcriptome signatures were similarly robust in kidney as compared to the lung, but more modest in spleen. Transcriptomic signatures in lung and kidney were indicative of infiltration and/or expansion of neutrophils, macrophages, dendritic cells, B cells, and T cells that corresponded with accelerated autoimmune pathogenesis. Taken together, airway exposure to cSiO2 elicited aberrant mRNA signatures for both innate and adaptive immunity that were consistent with establishment of the lung as the central autoimmune nexus for launching systemic autoimmunity and ultimately, kidney injury.

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.

Ectopic lymphoid structures (ELS) consist of B-cell and T-cell aggregates that are initiated de novo in inflamed tissues outside of secondary lymphoid organs. When organized within follicular dendritic cell (FDC) networks, ELS contain functional germinal centers that can yield autoantibody-secreting plasma cells and promote autoimmune disease. Intranasal instillation of lupus-prone mice with crystalline silica (cSiO2), a respirable particle linked to human lupus, triggers ELS formation in the lung, systemic autoantibodies, and early onset of glomerulonephritis. Here we tested the hypothesis that consumption of docosahexaenoic acid (DHA), an ω-3 polyunsaturated fatty acid with anti-inflammatory properties, influences the temporal profile of cSiO2-induced pulmonary ectopic germinal center formation and development of glomerulonephritis. Female NZBWF1 mice (6-wk old) were fed purified isocaloric diets supplemented with 0, 4, or 10 g/kg DHA - calorically equivalent to 0, 2, or 5 g DHA per day consumption by humans, respectively. Beginning at age 8 wk, mice were intranasally instilled with 1 mg cSiO2, or saline vehicle alone, once per wk, for 4 wk. Cohorts were sacrificed 1, 5, 9, or 13 wk post-instillation (PI) of the last cSiO2 dose, and lung and kidney lesions were investigated by histopathology. Tissue fatty acid analyses confirmed uniform dose-dependent DHA incorporation across all cohorts. As early as 1 wk PI, inflammation comprising of B (CD45R+) and T (CD3+) cell accumulation was observed in lungs of cSiO2-treated mice compared to vehicle controls; these responses intensified over time. Marked follicular dendritic cell (FDC; CD21+/CD35+) networking appeared at 9 and 13 wk PI. IgG+ plasma cells suggestive of mature germinal centers were evident at 13 wk. DHA supplementation dramatically suppressed cSiO2-triggered B-cell, T-cell, FDC, and IgG+ plasma cell appearance in the lungs as well as anti-dsDNA IgG in bronchial lavage fluid and plasma over the course of the experiment. cSiO2 induced glomerulonephritis with concomitant B-cell accumulation in the renal cortex at 13 wk PI but this response was abrogated by DHA feeding. Taken together, realistic dietary DHA supplementation prevented initiation and/or progression of ectopic lymphoid neogenesis, germinal center development, systemic autoantibody elevation, and resultant glomerulonephritis in this unique preclinical model of environment-triggered lupus.

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.

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.

Somatic cell NT (SCNT) efficiency remains poor, preventing the technology from being regularly used in the agricultural industry. It is believed that faulty epigenetic reprogramming of SCNT embryos leads to the low overall success. A clear apoptotic signature is associated with inappropriate gene expression and epigenomic aberrancies in many experimental cell culture systems, and we hypothesised that an apoptosis biomarker could be used to effectively separate properly reprogrammed porcine SCNT embryos from those that are destined to fail due to incomplete reprogramming. Therefore, our objective was to evaluate global gene expression and DNA methylation patterns in high- and low-apoptosis individual embryos in an effort to characterise the extent of genomic reprogramming that had taken place. Porcine SCNT blastocysts on Day 6 of development were stained with a nontoxic, noninvasive caspase activity reporter, and the top and bottom 20% of detected caspase activity were classified as high and low apoptosis, respectively (3 replicate cloning sessions; n=13 embryos per group). Genomic DNA and total RNA were isolated from each individual blastocyst. The RNA sequencing libraries were prepared using the Ovation SoLo RNA-Seq system (NuGen, San Carlos, CA, USA). Reduced representation bisulfite sequencing libraries were prepared for DNA methylation analysis using a modification of the single-cell reduced representation bisulfite sequencing global DNA methylation analysis approach detailed by Guo et al. (2015 Nat. Protoc. 10, 645-59). The RNA sequencing analysis using EdgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html) revealed 175 total differentially expressed genes (fold change ≥1.5; false discovery rate ≤0.05) between the high- and low-apoptosis SCNT embryos. This list of differentially expressed genes was used to perform enrichment analysis to identify overrepresented Gene Ontology (GO) terms or Kyoto Encyclopedia of Genes and Genomes pathways (DAVID Ease version 6.8 (https://david.ncifcrf.gov/) against the Sus scrofa background genome). However, no significantly enriched GO terms or pathways were identified (false discovery rate P>0.05). Analysis of global DNA methylation patterns between high- and low-apoptosis SCNT embryos using MethylKit (Akalin et al. 2012Genome Biol. 13, R87) revealed 335 differentially methylated 100-bp regions with at least 25% difference in methylation (adjusted P ≤ 0.01). Gene transcription start sites associated with these regions were used for enrichment analysis; again, no significant enrichment of GO terms or Kyoto Encyclopedia of Genes and Genomes pathways was identified. Principal component analysis of CpG methylation showed the low-apoptosis embryos clustering more tightly than the high-apoptosis embryos, which were highly scattered. Ongoing comparisons of high- and low-apoptosis cloned embryos with naturally fertilized embryos produced invivo may provide more information about which embryos were properly reprogrammed. Although we are still pursuing a link between reprogramming and gene expression in high- and low-apoptosis embryos, we conclude that these data support a model of stochastic epigenetic reprogramming following SCNT and reinforce the necessity of identifying embryos most likely to be successful due to proper epigenetic reprogramming in order to increase SCNT efficiency.

Catalytically active human flavin-containing monooxygenase isoform 2 (FMO2.1) is encoded by an allele detected only in individuals of African or Hispanic origin. Genotyping and haplotyping studies indicate that S195L and N413K occasionally occur secondary to the functional FMO2*1 allele encoding reference protein Gln472. Sulfoxygenation under a range of conditions reveals the role these alterations may play in individuals expressing active FMO2 and provides insight into FMO structure. Expressed S195L lost rather than gained activity as pH was increased or when cholate was present. The activity of S195L was mostly eliminated after heating at 45°C for 5 min in the absence of NADPH, but activity was preserved if NADPH was present. By contrast, Gln472 was less sensitive to heat, a response not affected by NADPH. A major consequence of the S195L mutation was a mean 12-fold increase in Km for NADPH compared with Gln472. Modeling an S213L substitution, the equivalent site, in the structural model of FMO from the Methylophaga bacterium leads to disruption of interactions with NADP+. N413K had the same pattern of activity as Gln472 in response to pH, cholate, and magnesium, but product formation was always elevated by comparison. N413K also lost more activity when heated than Gln472; however, NADPH attenuated this loss. The major effects of N413K were increases in velocity and kcat compared with Gln472. Although these allelic variants are expected to occur infrequently as mutations to the FMO2*1 allele, they contribute to our overall understanding of mammalian FMO structure and function.

Cancer risk assessment utilizing rodents requires extrapolation across five orders of magnitude to estimate the Virtually Safe Dose (VSD). Regulatory agencies rely upon the Linear Extrapolated Dose (LED) except when sufficient information on mechanism of action justifies alternative models. Rainbow trout (Oncorhynchus mykiss) has been utilized at Oregon State University as a model for human cancer for forty years. Low cost and high capacity, made possible by our unique facility, along with low spontaneous background and high sensitivity, allow design and conduct of statistically challenging studies not possible in rodents. Utilization of custom microarrays demonstrates similarities in gene expression in trout and human hepatocellular carcinoma (HCC). We have completed one study employing over 42,000 trout with dibenzo[a,l]pyrene (DBP) and determined the dose resulting in 1 additional cancer in 5000 animals, a 50-fold enhancement over the mouse ED(01) study. Liver tumor incidence at low dose deviated significantly from linearity (concave down), whereas, DBP-DNA adductions deviated slightly (convex up). A second study is underway with aflatoxin B(1) (AFB(1)). Results to date indicate AFB(1) at low dose, in contrast to DBP, elicits a linear dose-response function on the log-log scale which falls below the LED with a slope slightly greater than 1.0. Such studies demonstrate the statistical power of the trout cancer model and strengthen the case for incorporation of these data-sets into risk assessment for these environmental human carcinogens.

The cDNAs of the G protein-coupled receptor 54 (GPR54) and three prepro-gonadotropin-releasing hormones, GnRH-I (seabream GnRH), GnRH-II (chicken GnRH-II), and GnRH-III (salmon GnRH) were isolated and cloned from the brain of the teleost fish cobia, Rachycentron canadum. The cobia GPR54 cDNA was 95 and 51–56% identical to those of tilapia and mammalian models respectively. The GnRH cDNA sequences of cobia showed strong identities to those of tilapia, Atlantic croaker, red drum, and the seabass and seabream species. The real-time quantitative RT-PCR methods allowed detection of all three GnRH mRNAs on the first day after hatching (DAH). The GnRH-I mRNA levels, which were the lowest among the three GnRHs, increased gradually with two distinct peaks in larvae at 3 and 4 DAH. On the other hand, GnRH-II and GnRH-III mRNAs were significantly higher in larvae at 2 and 6 DAH compared with those on the preceding days. In addition, significant peaks of all the three GnRH mRNAs were observed in the brains of 26-day-old fish. The finding of higher GnRH-I and GnRH-II mRNAs in males than females at 153 DAH may be related to early puberty observed during the first year in laboratory-reared male cobia. Moreover, this study demonstrates for the first time the expression of GPR54 mRNA during larval development in a vertebrate species. The concomitant expression patterns of GPR54 and GnRH mRNAs during different stages of larval and juvenile developments, and during early puberty in male cobia suggest a potential relationship between GPR54 and multiple GnRHs during these stages of development consistent with the role of GPR54 in controlling GnRH release in mammals. The increase in GPR54 and GnRH mRNAs observed during early puberty in cobia is consistent with a similar change reported in pubertal rats. This finding together with the localization of GPR54 mRNAs on GnRH neurons in fish and mammals suggests that the GPR54–GnRH interactions may be conserved in different vertebrate groups.

The ontogeny of the digestive system of cobia Rachycentron canadum from hatching to 22 days post-hatch (dph) (20·1 mm standard length) was examined with light microscopy. The activities of selected pancreatic enzymes were also determined during this period in order to optimize current rearing methods for this species. At hatching (3·6 mm), the digestive tract consisted of a relatively undifferentiated, straight tube positioned dorsally to the yolk sac. The major morphological changes in the digestive tract primarily occurred over the first 1–4 dph (3·6–4·4 mm). During this time, larvae began exogenous feeding (3 dph) and the digestive tract differentiated into five histologically distinct regions: buccopharynx, oesophagus, stomach anlage, anterior intestine and posterior intestine. Yolk reserves were exhausted by 5 dph (4·5 mm) and the oil globule began rapidly decreasing in size disappearing entirely by 9–10 dph (6·3–6·8 mm). Gastric glands differentiated at this time, and by 12 dph (8·1 mm) surface mucous cells of the stomach anlage stained positive for neutral mucosubstances. By 16 dph (11·6 mm), the blind sac (fundic region) of the stomach formed as did the pyloric caecae which initially appeared as a single protrusion of the anterior intestine just ventral to the pyloric sphincter. Generally, enzyme activities (U larva−1) for amylase (0·0–1·8), chymotrypsin (0·0–7902·4), trypsin (0·2–16·6) and lipase (9·3–1319·0) were measurable at or soon after hatching and increased steadily from c. 8–22 dph (5·7–20·1 mm). The results of this study are discussed in terms of current and future weaning practices of this species.

Molecules that can be used to deliver a controlled amount of carbon monoxide (CO) have the potential to facilitate investigations into the roles of this gaseous molecule in biology and advance therapeutic treatments. This has led to the development of light-induced CO-releasing molecules (photoCORMs). A goal in this field of research is the development of molecules that exhibit a combination of controlled CO release, favorable biological properties (e.g., low toxicity and trackability in cells), and structural tunability to affect CO release. Herein, we report a new biologically-inspired organic photoCORM motif that exhibits several features that are desirable in a next-generation photoCORM. We show that 3-hydroxyflavone-based compounds are easily synthesized and modified to impart changes in absorption features and quantum yield for CO release, exhibit low toxicity, are trackable in cells, and can exhibit both O2-dependent and -independent CO release reactivity.

Our research team developed a new total Western diet (TWD) for rodents that mimics typical human nutrition patterns with respect to macronutrient composition (amounts and types of sugar, fat, prote...

Previous investigations in Atlantic croaker ovaries and primary co-cultured theca and granulosa cells have identified multiple signal transduction pathways involved in the control of gonadotropin-induced steroidogenesis, including adenylyl cyclase- and calcium-dependent signaling pathways. In the present study, evidence was obtained for an involvement of a third signal transduction pathway, a mitogen-activated protein kinase (MAP kinase) signaling cascade, in the regulation of gonadal steroidogenesis in this lower vertebrate teleost model. Gonadotropin-stimulated testosterone synthesis was markedly attenuated by two antagonists of mitogen-activated protein kinase kinases 1/2 (MEK1/2, also known as Map2k1/Map2k2). Moreover, treatment with gonadotropin-induced MEK1/2-dependent phosphorylation of extracellular signal-regulated protein kinases 1/2 (ERK1/2, also known as Mapk3/Mapk1) in a concentration- and time-dependent manner in co-cultured croaker theca and granulosa cells. Active MEK1/2 was required for a complete steroidogenic response to activators of the adenylyl cyclase pathway, including forskolin and dbcAMP, suggesting that the target(s) of MAP kinase signaling are distal to cAMP generation and activation of cAMP-dependent protein kinase (PKA). Interestingly, dbcAMP caused a similar increase of ERK1/2 phosphorylation as was observed with gonadotropin treatment, although an inhibitor of PKA did not attenuate this response. Finally, there was no evidence of cross-talk between calcium-dependent signaling pathways and this MAP kinase cascade. While drugs that block calcium-dependent signal transduction, including inhibitors of voltage-sensitive calcium channels, calmodulin, and calcium/calmodulin-dependent kinases, significantly reduced gonadotropin-induced testosterone accumulation, these drugs had no apparent effect on hCG-induced ERK1/2 phosphorylation.

The identities of the membrane receptors mediating the majority of rapid, cell surface-initiated, nongenomic (i.e. nonclassical) steroid actions described to date are unclear. Two novel 7-transmembrane spanning proteins, representing two distinct classes of steroid membrane receptors, membrane progestin receptor alpha (mPRalpha) and a membrane estrogen receptor (mER), GPR30, have recently been identified in several vertebrate species. Evidence that both receptors activate G-proteins and function as G-protein coupled receptors (GPCRs) is briefly reviewed. New data on progestin actions on fish gametes suggest a widespread involvement of mPRalpha in oocyte maturation and sperm hyperactivity in this vertebrate group. Information on the second messenger pathways activated upon estrogen binding to a membrane estrogen receptor in croaker gonads and preliminary evidence for the presence of a GPR30-like protein in fish gonads are discussed. Finally, initial characterization of the ligand binding, G-protein activation and molecular size of a membrane androgen receptor (mAR) in croaker ovaries suggests the presence of a third unique steroid receptor in fish gonads that also may function as a GPCR.

The involvement of calcium-dependent signal transduction pathways in the regulation of ovarian steroidogenesis was investigated in Atlantic croaker. Treatment with the calcium ionophores A23187 and ionomycin caused a 2- to 5-fold increase in basal steroid accumulation by croaker ovarian tissue in vitro. A23187 potentiated human chorionic gonadotropin (hCG)-induced testosterone (T) accumulation, whereas it inhibited accumulation of estradiol-17beta (E(2)) and the conversion of T to E(2), suggesting that intracellular calcium modulates aromatase enzyme activity. Gonadotropin stimulation of ovarian steroidogenesis was decreased in the presence of EGTA and inhibitors of voltage-sensitive calcium channels (VSCCs) and inositol-1,4,5-triphosphate-receptors (IP(3)Rs), indicating that releases of calcium from both intracellular and extracellular stores are components of the signal transduction pathways initiated by gonadotropin. Calmodulin is also involved in the regulation of ovarian steroidogenesis in croaker, since the calmodulin inhibitors W-7 and trifluoperazine (TFP) attenuated hCG-stimulated T and E(2) accumulation. These results are broadly similar to those reported previously in goldfish and suggest that the major calcium-dependent signaling pathways involved in gonadotropin stimulation of ovarian steroidogenesis in tetrapods are also present in teleosts. In addition, the involvement of calcium in the regulation of aromatase activity was demonstrated for the first time in a vertebrate ovary. Finally, acute exposure to 0.001-1 ppm Aroclor 1254 induced up to a 5-fold increase in hCG-stimulated E(2) accumulation, and this effect was attenuated by co-treatment with inhibitors of VSCCs and calmodulin, suggesting the existence of a novel mechanism of endocrine disruption by an environmental contaminant involving alteration of calcium-dependent signaling pathways regulating steroidogenesis.

Background: Rodent models have been an invaluable resource for biomedical research and have been instrumental for countless advances in our understanding of biology and human disease. However, inherent to using these models is the issue of translatability of research findings to human populations. Some differences between humans and rodents can never be reconciled because of key differences in physiology. However, rodent models have evolved over time through innovations in genetics and standardized animal diets, resulting in reduced variability across experiments. Developing animal diets that more closely emulate what humans eat will help increase the translational fidelity of animal models to human populations. This review will focus on the role of basal laboratory diets for improving animal models. Keywords: laboratory rodent diets, total Western Diet, allometric scaling, nutrient density scaling

Previously, we used NHANES data to formulate the TWD, a rodent diet that emulates average American intake levels for macro and micronutrients using nutrient density. We have demonstrated that the T...

Traditionally, mouse humanization studies have used human fecal transfer to germ-free animals. This practice requires gnotobiotic facilities and is restricted to gnotobiotic mouse lines, which limits humanized mouse research. We have developed a generalizable method to humanize non germ-free mice using antibiotic treatment and human fecal transfer. The method involves depleting resident intestinal microbiota with broad-spectrum antibiotics, introducing human microbiota from frozen fecal samples by weekly gavage, and maintaining mice in HEPA-filtered microisolator cages. Pyrosequencing cecal microbiota 16S rRNA genes showed that recipient mice adopt a humanized microbiota profile analogous to their human donors, and distinct from mice treated with only antibiotics (no fecal transfer) or untreated control mice. In the humanized mice, 75% of the sequence mass was observed in their respective human donor and conversely, 68% of the donor sequence mass was recovered in the recipient mice. Principal component analyses of GC- and HPLC-separated cecal metabolites were performed to determine effects of transplanted microbiota on the metabolome. Cecal metabolite profiles of mice treated with only antibiotics (no fecal transfer) and control mice were dissimilar from each other and from humanized mice. Metabolite profiles for mice humanized from different donor samples clustered near each other, yet were sufficiently distinct that separate clusters were apparent for each donor. Also, cecal concentrations of 57 metabolites were significantly different between humanization treatments. These data demonstrate that our protocol can be used to humanize non germ-free mice and is sufficiently robust to generate metabolomic differences between mice humanized from different human donors.