Brief Summary of Minutes of Annual Meeting

The 2015 Annual Meeting of the W-3122 Multistate Group was called to order by the Chair, Dr. Abby Benninghoff on the morning of October 8.  Dr. Mike Harrington (WAAESD) provided an overview of the status of the multi-state program and current funding priorities and budget status at USDA NIFA.  Last year, this group was recognized as the Western outstanding multi-state group.  Dr. Harrington would like to resubmit a nomination for W3122 to be considered for the national award again this year.  He will request updated grant totals and publications in 2016 to prepare the nomination.  Each member present delivered a presentation to update the group on his/her progress (content summarized in the Annual Group Report).  At the business meeting, new officers for 2015-2016 were selected:  Chair, Nancy Turner; Vice-Chair, Meijun Zhu; and Secretary, Yuhua Farnell.  Also, the dates for the 2016 annual meeting will be October 6-7 and the meeting will be held in Portland, Oregon.

OBJECTIVE 1: Determine the mechanisms by which dietary bioactive compounds protect against human diseases.

Dr. Roger Coulombe and colleagues are studying protective hepatic glutathione S-transferases (GST), universal phase II enzymes that detoxify dietary and environmental toxins and carcinogens such as aflatoxin B1 (AFB1) in an avian susceptibility model.  Resistance to dietary carcinogens such as AFB₁ is strongly associated with stable expression of protective hepatic glutathione S-transferases (GST), universal phase II enzymes that detoxify dietary and environmental toxins and carcinogens such as aflatoxin B₁. Expression of GSTs is enhanced by certain dietary bioactives, such as those from Brassica vegetables. Unlike their more susceptible counterparts, wild turkeys possess functional hepatic GSTs with AFB₁ detoxification activity.  We expressed, cloned and functionally characterized six likely candidates: GSTA genes GSTA1.1, GSTA1.2, GSTA1.3, GSTA2, GSTA3, and GSTA4 from the livers of wild and domestic turkeys.  In contrast to their hepatic forms, all E. coli-expressed recombinant GSTAs from both domestic and wild turkeys, had AFBO-detoxification activity, implying that hepatic GSTs in domestic turkeys are downregulated by one or more genetic or epigenetic mechanisms.  Focused sequencing of the hepatic transcriptome revealed significantly more hepatic GSTAs are expressed in wild than domestic birds, a difference more marked for two genes - GSTA3 and GSTA4. As in isolated populations of people with cancer susceptibility due to GST polymorphisms, loss of protective GST alleles in domestic turkeys is the likely mechanism for their extreme sensitivity compared to wild birds. This year, we initiated large-scale miRNA discovery experiments to determine whether unique miRNAs can inhibit expression of GSTA mRNAs.

After RNA sequencing of wild and domestic livers, Dr. Coulombe’s group compiled a list of all known or predicted precursor miRNA sequences for turkey from RNACentral (179 sequences) and Ensembl (430 sequences) and filtered for duplicates. Mature miRNA sequences for these 590 unique precursor miRNAs were predicted using MatureBayes, yielding 950 unique mature miRNA sequences. BLAST databases of mRNA sequences from turkey transcriptomes were generated using RNA-seq data from our newly published data (Monson et al. 2015). The RNA-seq dataset includes transcripts of wild-type and domesticated-type turkeys treated with AFB1 and untreated controls. The Illumina reads from the four birds in each type-treatment were pooled and filtered for duplicates (while keeping track of the number of reads associated with each unique sequence). A separate BLAST database for each type-treatment was then generated from the unique RNA sequences.  We used StarMir to predict interaction sites (both seed-pairing and seedless interactions) between the predicted mature miRNAs corresponding to those 15 precursor miRNAs and the mRNA sequences for turkey GSTA3 and GSTA4. Seven mature miRNAs had predicted interactions (with probability >0.7) at sites with known SNPs between wild and domesticated mRNAs, making them candidates for further experimentation.  Some of the eight mature miRNAs corresponding to the four precursor miRNAs identified as putative candidates using the first method were also among the seven candidates identified using the second method, so between the two methods, a total of 13 unique mature miRNAs were identified as candidates for further study.

In work completed over the past year, Dr. Benninghoff’s research group (USU) aimed to determine whether dietary supplementation with tart cherries prevented colon tumor development in mice consuming a Western diet compared to a prudent diet.  They hypothesized that dietary supplementation with freeze-dried whole tart cherries would suppress development of colon tumors in a model of inflammation-associated colorectal cancer.  Mice were fed either AIN93G (optimized for rodent health) or the total Western diet (TWD), each with and without Montmorency tart cherry powder added to the diet for a total anthocyanin content of 188 mg/kg diet.  TWD consumption markedly enhanced colitis (40-fold increase in disease activity) and tumor multiplicity (near 6-fold increase) compared consumption of the optimal AIN93G diet.  Supplementation with tart cherry powder caused a significant 40% reduction in tumor incidence in mice fed AIN93G, whereas tart cherries had no effect on tumor incidence in mice fed TWD.  However, tart cherry powder supplementation did not significantly affect tumor multiplicity, tumor size or colitis in mice fed either basal diet.  The chemoprevention apparent in mice fed an optimal diet supplemented with tart cherry powder contrasted with a prior observation by Dr. Benninghoff’s group that green tea supplementation was effective at reducing development of aberrant colonic crypts, but only in mice fed TWD.  Thus, these observations point to important interactions between basal diets and dietary bioactive supplements and underscore the need for careful consideration of the role of basal diet in dietary chemoprevention studies in rodents.  

Previously, Dr. Benninghoff’s group reported that consumption of the TWD promoted tumorigenesis an inflammation-associated model of colorectal carcinogenesis.  This year, her team completed RNAseq analysis of colon mucosa obtained from mice fed either an optimal diet, a 45% fat diet or the TWD.  By assessing gene expression in sham mice, we were able to identify functional pathways altered by basal diet as opposed to pathways associated with carcinogenesis.  Importantly, the 45% fat diet had very little effect on gene expression in colon mucosa compared to mice fed the optimal AIN93G diet.  However, changes in biological processes associated with immune response were very evident in mice fed the TWD compared to both the AIN93G and 45% fat diets.  This observation suggests that consumption of the TWD alters immune function in the colon muocsa; however, a link between this observation and colon carcinogenesis was not considered in this study design.

Dr. Pratibha Nerurkar and colleagues (University of Hawaii) have studied the anti-diabetic properties of bitter mellon (noni) juice.  Currently, her group is focused on health disparities among enthic minorities.  Specifically, In Hawaii, minority populations such as Native Hawaiians and Pacific Islanders (NHPI) have more than twice the rate of obesity-associated type 2 diabetes (T2D), as compared to Caucasians and more than five times as likely to die from T2D. Current therapies for obesity are complicated due to factors including an inability to maintain long-term weight loss and drug-drug interactions. In addition, conventional therapies may not be affordable, suitable and/or acceptable for culturally sensitive minority populations. There is a growing awareness and mounting body of scientific evidence, that successful implementation of strategies to control T2D among ethnic minorities will require culturally appropriate interventions.  We have demonstrated that laboratory-prepared noni juice (fNJ) improves not only glucose and lipid metabolism, but also prevent weight gain in mice fed high-fat-diet (HFD) containing 58% fat, Although world wide market for noni products is currently estimated at $2 billion and laboratory-based research have supported health claims of noni, clinical studies supporting health benefits of noni are scan and limited to Tahitian noni juice. Current studies are planned to compare the anti-diabetic effects of fNJ and commercial noni juices and differences in chemical metabolites.

Dr. James Pestka (Michigan State University) and colleagues are investigating the impact of exposure to respiratory toxicants, such as silica, on risk for developing lupus, a debilitating autoimmune disease that adversely affects 1.5 million Americans. While the genome is a primary predisposing factor for autoimmunity, lifetime environmental exposures to factors (ie. exposome)  such as environmental toxicants and diet are now recognized to modulate hereditary effects. Experimental animal and epidemiological studies have linked exposure to the respiratory toxicant silica to lupus and other autoimmune diseases. Importantly, nearly 2 million Americans are occupationally exposed to respirable silica.  In contrast, both animal and clinical studies suggest that consumption of DHA and other ω-3 PUFAs can both prevent and resolve inflammation and autoimmune nephritis.  Little is known about how respiratory toxicants like silica trigger lupus or how DHA supplementation could be harnessed to block environmental triggering. This critical barrier results in unnecessary lupus burden in genetically predisposed people that are exposed to respiratory toxicants. Lupus onset/progression in such individuals could potentially be mitigated by development and implementation of effective, low cost prophylactic approaches employing optimized supplementation with ω-3 PUFAs, already regularly consumed by over 20 million Americans. Our research focuses on using such an approach to prevent lupus.

The initiating step in lupus is loss of tolerance to nuclear self-antigens resulting in production of autoreactive antibodies and formation of circulating immune complexes. These deposit in the kidney and other organs where they promote infiltration and activation of circulating mononuclear cells, mediating advanced glomerulonephritis that can culminate in end-stage renal failure.  In a recent investigation, Dr. Pestka’s group used female lupus-prone NZBWF1 mice to compare dietary effects on the onset of autoimmunity as reflected by autoantibody production and glomerulonephritis in animals consuming: 1) an n-3 PUFA-rich diet containing DHA-enriched fish oil, 2)  an n-6 PUFA-rich Western-style diet  containing corn oil or  3) an n-9 monounsaturated fatty acid (MUFA)-rich Mediterranean-style diet containing high oleic safflower oil. Consumption of n-3 PUFA DHA effectively blunted the elevated autoantibody and nephritic responses in this model that were observed at 34 wk of age in mice consuming either the Western-style or Mediterranean-style diets. Remarkably, these attenuating effects co-occurred with generalized downregulation of CD4+ T cell-associated genes in the kidney and, to a lesser extent,  in spleen that are associated with antigen presentation, T cell activation, leukocyte recruitment, B cell activation/differentiation and inflammatory responses. Many of these genes are under consideration as potential targets for development of expensive biological therapeutics for human lupus including monoclonal antibodies and receptor antagonists.

In study that was published this year, Dr. Pestka and coworkers tested the hypothesis that early repeated short-term silica exposure will modulate both latency and severity of lupus in the female NZBWF1 mouse.  Weekly intranasal exposure to silica (0.25 and 1.0 mg) for 4 wk beginning at 9 wk of age both reduced latency and increased intensity of glomerulonephritis. silica elicited robust inflammatory responses in the lungs as evidenced by extensive perivascular and peribronchial lymphoplasmacytic infiltration consisting of IgG-producing plasma cells, and CD45R+ and CD3+ lymphocytes (B and T lymphocytes, respectively) that were highly suggestive of ectopic lymphoid tissue (ELT). In addition, there were elevated concentrations of immunoglobulins and the cytokines MCP-1, TNF-α and IL-6 in bronchoalveolar lavage fluid (BALF). silica-associated kidney and lung effects paralleled dose-dependent elevations of  autoantibodies and proinflammatory cytokines in plasma. Taken together, silica-induced pulmonary inflammation and ectopic lymphoid neogenesis in the NZBWF1 mouse corresponded closely to systemic inflammatory and autoimmune responses as well as the early initiation of pathological outcomes in the kidney.  These findings suggest that following airway exposure to silica, in lupus-prone mice, the lung serves as a platform for triggering systemic autoimmunity and lupus nephritis.

Dr. Nancy Turner (Texas A&M University) previously demonstrated that inclusion of sorghum brans containing polyphenolic compounds in the diet influenced butyrate concentrations, suggesting that changes in the microbiota were occurring as a result of these dietary interventions. As part of a recent project, they aimed to characterize the microbiota during colitis, and ascertain if polyphenol-rich sorghum bran diets mitigate these effects (Ritchie et al., 2015a). Rats (n=80) were fed diets containing 6% fiber from cellulose, or Black (3-deoxyanthocyanins), Sumac (condensed tannins), or Hi Tannin black (both) sorghum bran. Inflammation was induced three times using 3% DSS for 48 hr (40 rats, 2 wk separation), and the microbiota characterized by pyrosequencing. The Firmicutes/Bacteroidetes ratio was higher in DSS-treated rats consuming the cellulose diet. Colonic injury negatively correlated with Firmicutes, Actinobacteria, Lactobacillales, and Lactobacillus and positively correlated with Unknown/Unclassified. After the second DSS exposure, richness was significantly lower in Sumac and Hi Tannin black. Following the third DSS treatment, Bacteroidales, Bacteroides, Clostridiales, Lactobacillales, and Lactobacillus were reduced, with no Clostridium identified. Diet significantly affected Bacteroidales, Bacteroides, Clostridiales and Lactobacillus after the second and third DSS treatment. Analysis of samples collected after the third DSS treatment demonstrated diet significantly affected all genuses, including Bacteroides and Lactobacillus, and diversity and richness increased. Sumac and Hi Tannin black DSS-treated rats had significantly higher richness compared to controls. These observations suggest that sorghum brans containing polyphenolic compounds may protect against alterations observed during colitis including reduced microbial diversity and richness, and dysbiosis of Firmicutes/Bacteroidetes.

 Another project for Dr. Turner’s group focused on identifying how diet and environment influences the colonic microbiota was completed and published this year (Ritchie et al., 2015b). The aim of this study was to characterize how high and low linear energy transfer (LET) radiation, microgravity, and elevated dietary iron affect colon microbiota (determined by 16S rDNA pyrosequencing) and colon function.  Three independent experiments were conducted to achieve these goals: 1) fractionated low LET γ radiation (¹³⁷Cs, 3 Gy, RAD), high Fe diet (IRON) (650 mg/kg diet), and a combination of low LET γ radiation and high Fe diet (IRON+RAD) in male Sprague-Dawley rats; 2) high LET ³⁸Si particle exposure (0.050 Gy), 1/6 G partial weight bearing (PWB), and a combination of high LET ³⁸Si particle exposure and PWB in female BalbC/ByJ mice; and 3) 13 d spaceflight in female C57BL/6 mice. Low LET radiation, IRON and spaceflight increased Bacteroidetes and decreased Firmicutes. RAD and IRON+RAD increased Lactobacillales and lowered Clostridiales compared to the control (CON) and IRON treatments. Low LET radiation, IRON, and spaceflight did not significantly affect diversity or richness, or elevate pathogenic genera. Spaceflight increased Clostridiales and decreased Lactobacillales, and similar trends were observed in the experiment using a ground-based model of microgravity, suggesting altered gravity might affect colonic microbiota. Although we noted no differences in colon epithelial injury or inflammation, spaceflight elevated TGFβ gene expression. Microbiota and mucosal characterization in these models is a first step in understanding the impact of the space environment on intestinal health.  Dr. Turner and colleagues are currently following up these observations by performing a controlled-land based experiment to determine the impact of high iron diets and microgravity on colon microbial populations and intestinal health.

Dr. Turner’s most recently published work was conducted to explore the mechanisms whereby the combination of fish oil and butyrate protects against colon cancer development in our experimentally induced colon cancer model (Hong et al., 2015).  We hypothesized that this combination protects against colon cancer initiation by decreasing cell proliferation and increasing differentiation and apoptosis through a p27^Kip1 mediated mechanism. Rats were provided diets of corn or fish oil, with/without butyrate, and terminated 12, 24 or 48 h post azoxymethane (AOM) injection. Proliferation (Ki-67), differentiation (Dolichos Biflorus Agglutinin), apoptosis (TUNEL) and p27^Kip1 (cell cycle mediator) were measured in the same cell within crypts in order to examine the coordination of cell cycle as a function of diet. DNA damage (N⁷-methylguanine) was determined by quantitative immunohistochemical analysis. Dietary fish oil decreased DNA damage by 19% (P=0.001) and proliferation by 50% (P=0.003) and increased differentiation by 56% (P=0.039) compared to corn oil. When combined with butyrate, fish oil enhanced apoptosis 24 h post AOM injection compared to a corn oil/butyrate diet (P=0.039). There was an inverse relationship between crypt height and apoptosis in fish oil/butyrate group (r= -0.53, P=0.040). Corn oil/butyrate group showed a positive correlation between p27^Kip1 expression and proliferation (r= 0.61, P=0.035). These results indicate the in vivo effect of butyrate on apoptosis and proliferation is dependent on dietary lipid source. These results demonstrate the presence of an early coordinated colonocyte response by which fish oil and butyrate protects against colon tumorigenesis.

Dr. Turner continues to work towards understanding the impact of sorghum bran bioactive compounds on circulating metabolites in overweight subjects.  Results from their untargeted metabolomics analyses suggested there was a possibility of changes in lipoprotein metabolism.  As a result, they worked with Dr. Walzem to perform a detailed lipoprotein fraction analysis.  From this work they identified changes in three of the LDL subfractions and one of the HDL subfractions after our subjects consumed the low or high level of sumac sorghum cereal.  The mixture of changes detected was suggestive of a reduced risk of cardiovascular disease in obese subjects. 

Finally, Dr. Turner and colleagues completed an experiment to determine the impact of dried plums, a fiber and bioactive compound rich food source, on colon carcinogenesis.  They discovered that including the dried plums in the diet at a level that would correspond to the human equivalent serving size effectively reduced the formation of early preneoplastic lesions by 50%.  We detected significant changes in the distal colon microbiota of rats consuming the control diet.  However, rats consuming the dried plum diet exhibited minimal changes in the microbial populations between the proximal and distal colon, and the data indicated that changes in colon microbiota induced by the carcinogen were not as prominent in the dried plum group, when compared to the control diet group.  They are following up this project by performing untargeted metabolomics analyses on feces collected from the proximal and distal colon to determine if the changes in microbiota resulted in altered metabolite profiles, which may help explain suppression of colon tumorigenesis in those rats.  In addition, Dr. Turner is now collaborating with Dr. Ruth Globus who is determining the impact of dried plums on radiation damage to bones.  They are acquiring feces and colon tissue samples from those animals and will be determining if this dietary intervention serves to reduce the impact of radiation on colon health and microbial populations.

Dr. Tiffany Weir’s laboratory (Colorado State University) continued to explore the relationship between bioactive dietary components and their metabolism by gut bacteria as well as how they effect gut microbial composition for disease prevention. Recent research, including a human study conducted by the Weir laboratory, has shown that stool microbes and microbial metabolites differ between healthy individuals and those with colorectal cancer (CRC) (Weir et al., PLoS One 2013).  In a healthy human cohort, they demonstrated the feasibility of increasing dietary rice bran consumption for CRC prevention. Rice bran has a number of phytochemical components that contribute to reported anti-inflammatory and anti-carcinogenic bioactivities and could be utilized as a dietary component for chemoprevention. Dr. Weir and her colleagues showed that, in a healthy population, incorporation of dietary rice bran does not cause a large-scale disturbance in the intestinal ecosystem, but may induce subtle changes to the microbiota and metabolites detected in stool samples. The changes that they observed included increases in certain beneficial commensal bacteria and the introduction of bioactive metabolites (both through the diet and through microbial metabolism) that may reduce inflammation and contribute to CRC chemoprevention (Sheflin et al., Nutrients 2015). Further examination of the microbial changes introduced by rice bran consumption in a cohort of CRC survivors showed that there was increased production of the SCFA acetate by gut bacteria. Acetate is important in regulation of glucose metabolism and lipogenesis, modulating intestinal transit, and appetite regulation through interaction with intestinal G-coupled protein receptors, GPR41 and GPR43 in the intestinal epithelium and adipose tissue. Results of microbial sequencing in stool suggest transient increases in Bacteroides ovatus, which is a known butyrate producer, as well as in total bacterial diversity. Bacterial diversity has been shown to be inversely correlated with CRC occurrence. However, CRP levels were significantly increased in individuals consuming rice bran, so the actual benefits of RB consumption in terms of modulating intestinal inflammation need further exploration. Finally, examination of individual participant changes to microbiota suggest that there were responders and non-responders to the diet intervention and further work is needed to determine the factors involved in predicting responses. Metabolite data from pilot study healthy individuals has allowed us to refine rice bran-specific metabolite targets and the levels of these metabolites is currently being explored in stool.

Other ongoing projects by Dr. Weir’s group include examining the effects of caffeine on DNA repair mechanisms in enteric pathogens, identifying biomarkers in urine and stool that are consistent with fruit and vegetable intake levels as well as increased phytochemical consumption of intestinal bacteria composition and function, determining ability of fuzhuan tea metabolites to regulate cholesterol metabolism pathways in both preventive and reversal animal models, and identifying the ability of hops phytoestrogens to mitigate microbial dysbiosis and altered metabolism resulting from loss of estrogen.

Dr. Nicholas Bello and colleagues (Rutgers University) studied a ketone [4-(4-hydroxyphenyl)-2-butanone] derived from red raspberry (Rubus idaeus), which has purported antiobesity properties. Despite the popular press coverage of this proclaimed “miracle fat burner in a bottle” and huge internet sales, the safety, efficacy, and mechanism of action of raspberry ketone as a weight loss supplement are still unclear. While several studies have demonstrated lipolytic activity for raspberry ketone in vitro, there are only two in vivo studies in rodents that have examined the ability of raspberry ketone to prevent obesity and reduce dyslipidemia. The purpose of these set of experiments was to determine whether raspberry ketone suppressed feeding behavior and whether the compound activated feeding-related neural pathways. They determined that raspberry ketone provided via oral gavage at 200mg/kg bw significantly decreased food intake by about 20% compared to mice dosed with 100 mg/kg or provided vehicle only, and that the suppression was evident from 0.5 to 24 hours post dosing.  The effect of raspberry ketone was apparently not linked to gastrointestinal illness. In order to determine whether raspberry ketone acts on the central controls of feeding, Dr. Bellow and coworkers examined the neural activation by c-Fos immunoreactivity following acute dosing (200 mg/kg) in the hindbrain and hypothalamus. They found a significant increase in the number of c-Fos positive cell in the nucleus of the solitary tract (NTS) in mice that received raspberry ketone compared to controls. In the arcuate nucleus of the hypothalamus, there were no differences in the number of positive cells between treatments.    

Dr. Richard Eisenstein (University of Wisconsin, Madison) and coworkers are determining the extent to which diet-induced obesity and iron overload activate similar signaling pathways that contribute to the individual disease etiology.  One goal of their work to understand the mechanistic basis of the mild anemia observed in obesity.  A related goal is on the so-called unfolded protein response (UPR) and signaling pathways down-stream of the UPR that may modulate production of the iron regulatory protein hepcidin.  Dr. Eisenstein’s group is currently focused on the diet-induced obesity aspect of this work.  It is well accepted that this model leads to activation of the UPR and we will be examining, in samples obtained in the last year, if UPR components such as the protein kinase PERK, a endoribonuclease IRE1 and the transcription factor XBP1 are activated.  They are also examining the iron status of the control and obese mice in order to relate activation of the UPR to changes in iron metabolism.  A key component of this is the liver derived peptide hepcidin which controls dietary iron absorption and iron recycling from old red cells by macrophages.  Evidence in the literature indicates that hepcidin expression is activated by the UPR. Activation of the UPR in obese subjects could stimulate hepcidin production which is predicted to reduce dietary iron absorption and iron recycling leading to anemia.  A final component of this work is to examine if expression of the transcription factor HIF2-alpha is altered in obese mice.  HIF2alpha expression is controlled at the translational level by iron and we hypothesis the UPR.  Some evidence suggests that HIF2alpha may control transcription of the hepcidin gene.  Our current study will allow us to examine if there is activation of the UPR in obese mice is associated with translational dysregulation of HIF2alpha mRNA and alterations in hepcidin gene expression.

Dr. Eisenstein and colleagues are also examining how the iron regulated RNA binding protein, iron regulatory protein 1 (IRP1), controls dietary iron absorption and erythropoiesis during early postnatal development.  An ultimate goal of this, and a subject of our recently submitted Hatch renewal proposal, is whether dietary factors known to control HIF2alpha expression might consequently control dietary iron absorption.  IRP1 is the key iron mediator of HIF2alpha mRNA translation.  IRP1 is a repressor of HIF2alpha synthesis and loss of IRP1 translationally activates HIF2alpha mRNA.  HIF2alpha is a transcriptional activator of the main blood forming hormone erythropoietin and also the iron transport system controlling dietary iron acquisition in the duodenum.  Mice lacking IRP1 develop a severe but transient polycythemia (too many red cells) and absorb more iron to promote red cell overproduction.  They recently found that these mice have very strongly repressed hepcidin mRNA at birth and we are examining whether this involves canonical or non-canonical (HIF2alpha) mechanisms for controlling hepcidin gene transcription.  In addition, they are currently determining the extent to which HIF2alpha-targeted intestinal iron transport genes are altered in neonatal mice lacking IRP1.  These studies will expand the understanding as to how dietary iron absorption is controlled during the neonatal period with a specific focus on how the efficiency of iron absorption may be controlled to limit development of iron deficiency and prevent over-absorption of iron in common iron-loading disorders.  Dr. Eisenstein is also examining the iron-dependent mechanisms controlling IRP1 function as a means to understand how dietary iron absorption is controlled in disease states including iron deficiency as well as in inflammatory disorders.   This includes the known impact of inflammatory scenarios in reducing intestinal iron absorption.  The ability of IRP1 to bind mRNA is determined by insertion or loss of an Fe-S cluster with the cluster-free apoprotein form binding RNA. A second mechanism involves iron-dependent degradation of the IRP1 apoprotein.  To better understand how dietary iron absorption if controlled we are focusing on the ability of IRP1 to be regulated by the Fe-S cluster vs. the protein degradation mechanism.  A manuscript on this work is in preparation.

Dr. Mendel Friedman (USDA-ARS Albany) showed that the tomato glycoalkaloid α-tomatine induces cell death in mouse colon cancer ct-26 cells and transplanted tumors in mice.  Tomatoes (Solanum lycopersicum) produce the bioactive glycoalkaloid α-tomatine. This study determined the effect of commercial α-tomatine on CT-26 colon cancer cells in vitro and in vivo in an intracutaneously transplanted mouse tumor. Cytotoxicity experiments showed that α-tomatine induces about 50% lysis of the colon cancer cells at 3.5 μM after 24 h of treatment. Intraperitoneally administered α-tomatine (5 mg/kg body weight) also markedly inhibited growth of the tumor using CT-26 cancer cells without causing body and organ weight changes. The reduced tumor growth in the mice by 38% after 2 weeks was the result of increased caspase-independent apoptosis associated with increased nuclear translocation of apoptoosis-inducing mitochondrial protein (AIF) and decreased survivin expression in tumor tissues. α-Tomatine in pure form and in tomatine-rich green tomatoes might prevent colon cancer.

 Dr. Friedman also investigated the effect of allyl isothiocyanate on developmental toxicity in exposed Xenopus laevis embryos. The pungent natural compound allyl isothiocyanate isolated from the seeds of Cruciferous (Brassica) plants such as mustard is reported to exhibit numerous beneficial health-promoting antimicrobial, antifungal, anticarcinogenic, cardioprotective, and neuroprotective properties. Because it is also reported to damage DNA and is toxic to aquatic organisms, the objective of the present study was to determine whether it possesses teratogenic properties. The frog embryo teratogenesis assay-.Xenopus (FETAX) was used to determine the following measures of developmental toxicity of the allyl isothiocyanate: (a) 96-h LC50, defined as the median concentration causing 50% embryo lethality; (b) 96-h EC50, defined as the median concentration causing 50% malformations of the surviving embryos; and (c) teratogenic malformation index (TI), equal to 96-h LC50/96-h EC50. The quantitative results and the photographs of embryos before and after exposure suggest that allyl isothiocyanate seems to exhibit moderate teratogenic properties.

 Dr. David Pagliarini at the Morgridge Institute for Research (Madison, WI) has have continued to investigate the effects of iron deprivation on the cellular control of mitochondrial biogenesis. His group previously discovered, using microarray and quantitative mass-spectrometry approaches, that depriving C2C12 mouse myotubes of iron, through treatment with the iron chelator deferoxamine (DFO) leads to a global decrease in the transcript abundance of mitochondrial- and nuclear-encoded mitochondrial genes and mitochondrial proteins. They found that this response to iron chelation is universal across a broad range of cell types, rapid (occurring within 24 hours) and dose-dependent. Additionally, they discovered that the effect on cellular gene expression and respiratory capacity can be fully reversed upon the reintroduction of iron, indicating that the response to iron deprivation is an adaptive cellular response rather than irreversible cellular damage. Lastly, they showed that this process is independent of well-established regulators of mitochondrial biogenesis, including PGC-1α, PGC-1β and HIF-1α. These results were published in the 2013 January issue of Cell Reports.

Following this publication, Dr. Pagliarini and colleagues continued their strategy of using large-scale, unbiased genomic and proteomic analyses in combination with targeted, hypothesis driven experiments to elucidate the molecular basis of this adaptive cellular response. First, they performed detailed timecourse measurements of mitochondrial transcript and protein levels following iron deprivation, quantified using RT-qPCR and immunoblotting respectively. From this timecourse, they found that the decrease in OxPhos (oxidative phosphorylation) proteins preceded the decrease in their corresponding transcripts. Additionally, they discovered that for the few OxPhos proteins that are unaffected by our iron chelation treatment (complexes III and V), their transcripts were nonetheless reduced (and that this observation is supported by our original microarray and mass spectrometry analyses). These results suggest that either the changes in mitochondrial transcript and protein levels occur though two separate mechanisms or that the changes in transcript abundance are in response to the protein changes, possibly through a retrograde signaling event. Next, Dr. Pagliarini and colleagues determined the level of gene expression at which iron deprivation operates (i.e., transcription or mRNA turnover) to cause the observed decrease in mitochondrial transcripts. To do so, they utilized RNA pulse-chase and labeling approaches, and from these analyses, determined that iron deprivation affects the synthesis of new OxPhos-encoding transcripts but has no measurable effect on transcript turnover.

Further, using a MS-based quantitative proteomics approach in collaboration with Professor John Denu’s lab (Biomolecular Chemistry, UW-Madison), Dr. Pagliarini and coworkers discovered that iron chelation causes dynamic changes in histone acetylation and methylation (including an overall decrease in histone acetylation) and have validated these observations via immunoblotting of histone extracts. They also found that the effect of iron chelation on transcript levels is fully reversed with (histone deacetylase) HDAC inhibition, consistent with histone post-translational modification (PTM)-mediated regulation of gene expression. To determine if changes in these modifications are found at nuclear-encoded mitochondrial genes whose expression is affected by iron deprivation, they performed chromatin immunoprecipitation (ChIP)-qPCR analyses. From these experiments, they observed reduced levels of histone acetylation at all mitochondrial genes analyzed, indicating a direct regulatory link between a decrease in histone acetylation and mitochondrial gene expression in response to iron deprivation.

In addition to the experiments above, Dr. Pagliarini’s group utilized both microarray and quantitative mass spectrometry approaches to identify potential novel protein regulators involved in the iron response. As expression changes in potential regulators of the response are likely to occur before the changes in mitochondrial gene expression, we collected samples for these analyses at an early time point (12 hours) following DFO treatment, prior to any observable mitochondrial change. They are currently performing loss- and gain-of-function analyses on potential regulators identified from these experiments. Using a candidate-based approach, we investigated the role of the major mitochondrial quality control proteases (Lon, HtrA2, ClpXP, i-AAA, m-AAA) in the observed response using RNAi and CRISPR/Cas9-mediated gene silencing. From this analysis we found that knockdown of these proteases did not affect the decrease in abundance of mitochondrial proteins following iron chelation. Last, they tested whether the mechanistic/mammalian target of rapamycin (mTOR), a protein kinase that can regulate mitochondrial biogenesis and through multiple mechanisms, is involved in the iron response. Surprisingly, treatment of cells mTOR inhibitors did not cause a decrease in OxPhos proteins and DFO treatment did not cause any changes in phosphorylation of the mTOR targets. Moreover, when cells were treated simultaneously with DFO and mTOR inhibitors, DFO caused the same decreases in OxPhos proteins, suggesting that the effect of iron deprivation on mitochondrial proteins is independent of mTOR activity and its downstream regulatory processes.

In Dr. David Williams’ laboratory (Oregon State University), the focus of this research continues to be on dietary chemoprevention of cancer, specifically protection of the fetus/infant from transplacental carcinogens by dietary supplementation with plant phytochemicals or the whole foods from which they were derived.  The emphasis continues to be on phytochemicals from cruciferous vegetables primarily indole-3-carbinol (I3C) and sulforaphane (SFN).  Dr. Williams and colleagues have recently explored the potential for I3C and SFN, added to the maternal diet during pregnancy, to protect the fetus/infant from transplacental cancer caused by an important class of environmental chemical pollutants, the polycyclic aromatic hydrocarbons (PAHs).  It has been estimated that 95% of exposure (for a non-smoker) to carcinogenic PAHs is through diet.  We found epigenetic markers such as DNA methylation, histone acetylation and profiles of non-coding RNAs were markedly altered in newborns from mothers exposed to a carcinogenic PAH in her diet and these alterations in the epigenome could be partially ameliorated by maternal dietary I3C.

OBJECTIVE 2. Elucidate mechanisms of action of dietary toxicants and develop biomarkers for human risk assessment and disease prevention.

Dr. William Helferich and colleagues (University of Illinois) developed a breast cancer metastasis model in which mammary tumor cells are injected into the marrow cavity of mice and metastasis to lung is followed by bioluminescent imaging (BLI).  This model can be used to evaluate pharmacological and dietary interventions to suppress cancer metastasis.  Breast cancer (BC) is the leading cancer in women worldwide. Metastasis occurs in stage IV BC with bone and lung being common metastatic sites. Bone is one of the most common sites for metastasis in breast cancer (BC). Micrometastasis in bone marrow was detected in 30% of patients with stage I, II, or III BC at time of primary surgery and is a strong indicator of poor prognosis. Aromatase inhibitors (AI) like letrozole suppress the conversion of androgens to estrogens and inhibit estrogen-responsive mammary tumor growth. The goal of this study was to evaluate the effects of letrozole on BC micro-metastatic tumor growth in bone and lung metastasis in intact and ovariectomized (OVX) mice with murine estrogen receptor negative (ER-) BC cells inoculated in tibia. Forty-eight BALB/c mice were randomly assigned to one of four groups: OVX, OVX+Letrozole, Intact, and Intact+Letrozole, and injected with 4T1 cells intra-tibially. Letrozole was subcutaneously injected daily for 23 days at a dose of 1.75 µg/g body weight. Tumor progression was monitored by bioluminescence imaging. OVX mice had lower serum estradiol than intact mice and greater tumor area and integrated density in the inoculated limb on D14 and D17. Letrozole decreased serum estradiol levels and reduced lung surface tumor numbers in intact animals. Mice receiving letrozole had significantly fewer tumor colonies and fewer proliferative cells in the lung than OVX and intact controls based on H&E and Ki-67 staining, respectively. In conclusion, tumors were larger with greater integrated density in inoculated limbs of OVX animals and letrozole reduced BC metastases to lungs, suggesting that though 4T1 cells are considered estrogen unresponsive, by lowering systemic estrogen level and possibly through interacting with the host organ, breast cancer metastasis to the lung was still effectively reduced by the aromatase inhibitor letrozole. Therefore, Letrozole may reduce bone micrometastasis to lung in the preclinical model and further investigations are needed before any recommendations are made to BC survivors.

Deoxynivalenol (DON or "vomitoxin"), a trichothecene mycotoxin produced by Fusarium, is a global food safety concern because it commonly contaminates cereal grains and has the potential to cause growth suppression and gastrointestinal disease in humans. Climate change, modified agricultural practices and recent globalization of trade in agricultural plants have increased Fusarium cereal blight, thereby greatly increasing grain contamination by DON and markedly expanding the contaminant profile to include other structurally-related 8-ketotrichothecenes (3-ADON, 15-ADON, nivalenol, fusarenon X) well as plant glucosidic metabolites such as DON-3-glucoside. Existing data are insufficient to predict the toxicity risks from exposure to mixtures in these emerging trichothecenes. Dr. Pestka and colleagues (MSU) tested the hypothesis that toxic equivalency factors (TEFs) for the 8-ketotrichothecenes derived from DON toxicity models will be applicable to food safety risk assessment and toxicity analysis.  This is being achieved in three ways. First, they compared the anorectic potencies of the the 8-ketotrichothecenes relative to DON using a mouse food refusal bioassay and relating this to the aberrant secretion of the gastrointestinal hormones peptide YY (PYY) and cholecystokinin (CCK). Second, they are ascertaining the comparative emetic potencies of 8-ketotrichothecenes relative to DON using a mink vomiting bioassay and are relating these data to the elevated secretion of gastrointestinal hormone PYY as well as serotonin. Finally, they are applying this knowledge by developing simple in vitro assay that will enable measurement of the trichothecene toxic equivalents in food samples for use as an intervention tool. Toward this end, several candidate enteroendocrine cell lines and endpoints (calcium mobilization, hormone secretion) have been identified.

Dr. Ron Riley (USDA-ARS Athens, GA)  investigated histone deacetylase inhibition in mouse embryonic fibroblasts from a fumonisin NTD-succeptible mouse model.  Studies have been completed showing that the target of the fumonisin-induced elevation in sphingoid base 1-phosphates and the sphingosine 1-phosphate analog FTY720-P is histone deacetylase. This is important because human exposure to histone deacetylase inhibitors during pregnancy causes neural tube defects (NTDs) in mice and both FTY720 and fumonisin B1 cause NTDs in mice.  Accumulation of nuclear Sa 1-P, reduction in histone deacetylase activity, and increased acetylation of specific histones represents a plausible mechanism for fumonisin exposure as a risk factor for NTDs in humans.  The information from in vivo mouse studies, in vitro studies in mouse, and human neural progenitor cells, and the finding that consumption of corn contaminated with high levels of fumonisin B1 is correlated with elevated sphinganine 1-phosphate in Guatemalan women, a country with high incidence of NTD, provides evidence that supports the hypothesis that consumption of corn-based diets containing high levels of fumonisin B1 could be a possible risk factor for NTDs in humans.  Dr. Riley also conducted metabolism studies of fumonisin B1 in rats.  A manuscript describing for the first time the in vivo metabolism of fumonisin B1 to ceramide like compounds has been published. The findings show that bioavailability of fumonisin B1 is higher than previously shown with significant amounts of the metabolites, relative to parent compound, accumulating in liver.  Characterizing metabolism and the biological activity(ies) of the metabolites is needed to better understand mechanisms of action and improve risk assessments. 

Dr. Riley and colleagues also determined that fumonisin exposure women was linked to inhibition of an enzyme that is a key event in farm and laboratory animal diseases.  Fumonisin B₁ (FB₁) is a toxic chemical produced by molds.  The molds that produce fumonisin are common in corn.  Consumption of contaminated corn by farm animals has been shown to be the cause of disease.  Fumonisin has been hypothesized to be an environmental risk factor for diseases in humans in countries where corn is a dietary staple and infection with the mold is likely. In order to determine if fumonisin contributes to disease in humans, methods were developed to measure changes in the urine and blood levels of chemicals that are indicators of changes indicative of pre-disease states in animal studies.  The human studies have focused on populations in Guatemala where corn is a dietary staple.  Intake of fumonisin in these populations can be very high.  Corn, urine and blood were sampled from over 1500 women and the results show that fumonisin intake and changes in a unique class of fats (sphingoid base 1-phosphates) in the blood are correlated in a manner that mimics the effects of fumonisin in laboratory animals.  The findings are consistent with the hypothesis that fumonisin inhibits the same enzyme in humans as it does in farm and laboratory animals consuming diets high in fumonisin.  These findings are the basis for development of biomarker-based studies in humans designed to identify possible human diseases where fumonisin could be a contributing factor and will provide an incentive to reduce fumonisin exposure in developing countries where corn is a dietary staple. These studies were conducted as part of a collaboration between USDA-TMRU (Athens, GA), Centro de Investigaciones en Nutrición y Salud in Guatemala, Creighton University and Duke University.  Additional human fumonisin biomarker-based studies are in progress in collaboration with Oakland University, Centro de Investigaciones en Nutrición y Salud (CIENSA), Michigan State University, and the University of Georgia.   The Oakland University study is focused in the Department of Huehuetenango (Guatemala), the CIENSA study is focused on the Departments of Guatemala and Alta Verapaz (Guatemala), the Michigan State University study is focused on Nepal and Tanzania, and the University of Georgia study is focused in Texas. The study in Huehuetenango is near completion and the results provide additional evidence for a link between fumonisin exposure and disruption of sphingolipid metabolism in humans.

Dr. David Williams’ group currently focuses on mechanisms of action and biomarkers associated with an important class as (3 of the top 10 ATSDR (Agency for Toxic Substances Disease Registry, a component of the CDC) environmental chemicals of concern are PAHs.  These have included molecular biomarkers such as alterations in levels of tumor suppressor genes, DNA adductions, etc., in addition to biological endpoints. Our group has been the first to utilize accelerator mass spectrometry (Lawrence Livermore National Laboratory, LLNL) to follow the uptake and elimination of PAHs at an environmentally relevant level of exposure (10% or less of average daily exposure).  Using this technology, we can measure femto(10^-15) grams of PAH/ml of blood. Ten femtograms/ml of blood is equivalent to 1 drop of water in 4000 Olympic-sized swimming pools.

OBJECTIVE 3. Discover and characterize novel bioactive dietary compounds that have beneficial or adverse effects on human health.

Dr. Benninghoff’s group (USU) completed a small pilot study initiated by a local high school student with an interest in dietary interventions for treatment of diabetes, specifically a Mexican folk remedy of incorporating canary seed into the diet to reduce glucose levels and improve insulin resistance. They tested the efficacy of canary seed supplementation for prevention of diet-induced glucose intolerance in mice fed a normal diet, a 60% high fat diet (to induce metabolic syndrome) and a high fat diet + 1% canary seed (CS). As expected, mice fed the high fat and high fat+canary seed diet experienced increased weight gain, primarily as excess fat. However, addition of canary seed to the high fat diet did not prevent excess weight gain or ameliorate the symptoms of diet-induced diabetes, as determined by elevated fasting glucose levels and glucose intolerance.  These observations contradict those reported previously by Perez Gutierrez (2014, Evidence-Based Complementary and Alternative Medicine, article ID 145901), although the experimental models were notably different; specifically, the current experiment evaluated CS as a preventative agent, while the Perez Gutierrez study used a therapeutic approach.

Dr. Michael Denison (UC Davis) and colleagues have continued to develop new and improved recombinant cell-based CALUX (chemically-activated luciferase expression) bioassays for screening of chemicals and extracts in order to detect and identify natural and synthetic activator/inhibitors of the Ah receptor (AhR) or steroid hormone receptors (i.e. estrogen receptor (ER)).  They successfully used these bioassay systems for the detection and characterization of food and herbal extracts for these chemicals, but these assays are not optimal in several respects and need improvement to expand and enhance their application for extracts of food and environmental samples.  Over the past year, they developed two improved CALUX assays for detecting agonists and antagonists for the AhR or ER that will greatly facilitate the detection and identification of chemicals that can interact with these receptor systems.  They previously developed an estrogen-responsive recombinant human ovarian (BG1Luc4E2) cell line recently accepted by the US Environmental Protection Agency (USEPA) and Organization for Economic Co-operation and Development (OECD) as an officially accepted bioanalytical method to detect agonists/antagonists. Unfortunately, these cells appear to contain only one of the two known ER isoforms, ERa but not ERb, and the differential ligand selectivity of these ERs indicates that the currently accepted screening method only detects a subset of total estrogenic chemicals. To improve the estrogen screening bioassay, BG1Luc4E2 cells were stably transfected with an ERb expression plasmid and positive clones identified using ERb-selective ligands (genistein and Br-ERb-041). A highly responsive clone (BG1LucERbc9) was identified that exhibited greater sensitivity and responsiveness to ERb-selective ligands than BG1Luc4E2 cells, and quantitative reverse-transcription polymerase chain reaction confirmed the presence of ERb expression in these cells. Screening of pesticides and industrial chemicals identified chemicals that preferentially stimulated ERb-dependent reporter gene expression. Together, the results of these studies not only demonstrate the utility of this new dual-ER recombinant cell line for detecting a broader range of estrogenic chemicals than the current BG1Luc4E2 cell line, but screening with both cell lines allows identification of ERa and ERb-selective chemicals.

In contrast, the AhR-responsive CALUX cell bioassay is commonly used for rapid screening of samples for the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), dioxin-like compounds, and AhR agonists/antagonists. This cell line has been used to detect such chemicals in a diverse array of matrices from environmental and biological samples, to food and commercial consumer products.  However, the limits of the lower sensitivity of this bioassay have restricted the application of this assay since even lower concentration of these compounds need to be detected.  Accordingly, we previously generated a novel third generation (G3) recombinant AhR-responsive mouse CALUX cell line (H1L7.5c3) with a significantly enhanced response to dioxin-like chemicals (DLCs) compared to existing AhR-CALUX cell bioassays. However, the elevated background luciferase activity of these cells and the absence of comparable G3 cell lines derived from other species (particularly that of human) have limited their utility for screening purposes. Thus, Dr. Denison’s group developed and characterized several new species-specific G3 recombinant AhR-responsive CALUX cell lines (human, rat and guinea pig) that exhibit significantly improved limit of detection and dramatically increased TCDD induction response. The low background luciferase activity, low minimal detection limit (0.1 pM TCDD) and enhanced induction response of the rat G3 cell line (H4L7.5c2) over the H1L7.5c3 mouse G3 cells, identifies them as a more optimal cell line for screening purposes. The utility of the new G3 CALUX cell lines were demonstrated by screening sediment extracts and a small chemical compound library for the presence of AhR agonists. The availability of new G3 CALUX cell lines with dramatically improved limit of detection and increased response will improve and enhance our ability to identify DLCs and AhR agonists/antagonists in a broader range of samples with low levels of contamination and/or in small sample volumes.

 Dr. Denison has also collaborated with numerous other investigators to demonstrate the presence of natural and synthetic DLCs and estrogenic chemicals in a variety of different matrices.  In addition, Malassezia furfur yeast strains isolated from human skin preferentially biosynthesize indole alkaloids which can be detected in human skin and are highly potent activators of the AhR and AhR-dependent gene expression. Chemical analysis of an EtOAc extract of a M. furfur strain obtained from diseased human skin and grown on L-tryptophan agar revealed several known AhR active tryptophan metabolites along with a previously unidentified compound, pityriazepin. While its structure resembled that of the known alkaloid pityriacitrin, the comprised pyridine ring had been transformed into an azepinone. The indoloazepinone scaffold of pityriazepin is extremely rare in nature and has only been reported once previously. Pityriazepin, like the other isolated compounds, was found to be a potent activator of the AhR-dependent reporter gene assays in recombinant cell lines derived from four different species, although significant species differences in relative potency was observed. The ability of pityriazepin to competitively bind to the AhR and directly stimulate AhR DNA binding classified it as a new naturally-occurring potent AhR agonist. Malassezia furfur produces an expanded collection of extremely potent naturally occurring AhR agonists, which produce their biological effects in a species-specific manner.

Dr. Mendel Friedman (USDA-ARS Albany) determined that rice hull liquid smoke protects fat mice against obesity. Obesity and being overweight are consequences of excessive adipogenesis.  In the United States, it is estimated that  about 80 million adult individuals are obese. In a collaborative study, we discovered that a rice hull liquid smoke extract exhibited strong anti-adipogenic effects in adipocytes (fat cells). The in vitro anti-obesity effect was confirmed in a 7-week mice feeding study. The extract prevented mice on the high-fat diet from gaining weight, caused significant reductions in total serum and liver cholesterol and triglyceride levels, and protected against liver injury, suggesting that the liquid smoke extract might inhibit obesity in humans. The liquid smoke extract, which seems to be safe for mice, could be added to different food categories (cheese, meat, seafood) using methods for the incorporation of wood-derived liquid smoke. US producers of liquid smoke should produce and market the rice hull smoke as a health-promoting food additive.

In other work, Dr. Friedman showed that rice hull smoke extract protects mice against a Salmonella lipopolysaccharide-induced endotoxemia.  Endotoxemia (sepsis, septic shock) is an inflammatory, virulent disease that results mainly from infection by Gram-negative bacteria. The present study investigates the inhibitory effects of a rice hull smoke extract (RHSE) against murine endotoxemia induced by Salmonella lipopolysaccharide and d-galactosamine (LPS/GalN). Pretreatment of the mice with RHSE via dietary administration for 2 weeks resulted in the suppression (in %) of LPS/GalN-induced catalase by 70.7, superoxide dismutase (SOD) by 54.6, and transaminase (GOT/GPT) liver enzymes by 40.6/62.5, the amelioration of necrotic liver lesions, and the reduction of tumor necrosis factor-α (TNF-α) by 61.1 and nitrite serum level by 83.4, as well as myeloperoxidase (MPO) enzyme associated with necrotic injury of the lung and kidney by 65.7 and 63.3, respectively. The RHSE also extended the lifespan of the toxemic mice. The results using inflammation biomarkers and from the lifespan studies suggest that the RHSE can protect mice against LPS/GalN-induced liver, lung, and kidney injuries and inflammation by blocking oxidative stress and TNF-α production, thereby increasing the survival of the toxic-shock-induced mice. It seems that the smoke extract also has the potential to serve as a new multifunctional resource in human food. 

OBJECTIVE 4. Increase beneficial or decrease adverse effects of bioactive constituents and microbes in food.

Dr. Mendel Friedman (USDA-ARS Albany) and colleagues determined that an elm tree bark bioprocessed in a liquid culture of mushroom mycelia protects mice against asthma. Asthma is a multifaceted chronic inflammatory disease of the lung airways, resulting in bronchial hyper-responsiveness following exposure to an allergen. There is no cure. Protein biomarkers can serve as a powerful detection tool in both clinical and basic research applications. The results of the present study with asthma-associated biomarkers (cytokines in serum and bronchoalveolar fluid, serum IgG and other biomarker levels, and inflammatory cell infiltration in the trachea and lungs) as well as RT-PCR and Western blot analysis of associated genes all show that the bioprocessed bark of the elm tree in a liquid mushroom mycelia culture normalized antigen-triggerred immune imbalance of the two T-helper proteins (Th1/Th2) in an mouse asthma model, suggesting that the treatment overcame  the manifestations of the numerous biomarkers associated with the asthmatic syndrome. The bioprocessed product was not toxic to leukemia cells, suggesting that it might be a safe product. The mouse assays demonstrate the potential value of the bioactive mushroom culture product as an anti-inflammatory and anti-allergic formulation that has the potential to prevent and/or treat allergic human diseases such as hay fever and asthma.

Dr. Friedman’s group also investigated antimicrobial activities of red wine-based formulations containing plant extracts against Escherichia coli O157: H7 and Salmonella enterica.  They evaluated the antimicrobial activities of three red wine based plant extract/plant essential oil (EO) formulations: olive extract powder/oregano EO, apple skin extract powder/lemongrass EO, and green tea extract powder/bitter almond EO, and their formula permutations against the foodborne pathogens Escherichia coli O157:H7 and Salmonella enterica. The formulations with oregano, lemongrass, or bitter almond EO inhibited the growth of both pathogens at acidic pH with bacterial loads of ~10⁴ or ~10⁹ bacteria/mL. At neutral pH, the formulations were less effective. Of the tested formulations, the lemongrass essential oil formulations, at pH 3.6, appeared to be the most effective against the tested pathogens, especially against Salmonella. The most active formulations merit evaluation for antimicrobial efficacy in liquid and solid food.

Dr. Mingruo Guo and colleagues (University of Vermont) investigated the effect of gamma radiation on the microbiological and physicochemical properties, protein-protein interaction and microstructure of whey proteins. Previous studies by the researchers have shown that whey protein structures can be modified by thermal and other chemical means to improve their functionality for use in value-added applications, such as wood and paper adhesives and stick glues. However, whey proteins can be aggregated and gelled prematurely when thermally treated and then stored at room temperature. Native protein structures in solutions and functional properties start to change when subjected to temperature up to 60°C and irreversible changes occur at higher temperatures above 70°C. Viscosity, turbidity, solubility, polymerization and other physicochemical changes of protein solutions can occur from the denaturation of whey proteins. The team treated samples of whey protein isolate (WPI) solutions (10-36% protein) with different dosages (10-35 KGy) of gamma radiation. They found that the lowest dosage (10 KGy) of gamma radiation used was sufficient to eliminate the presence of all viable microorganisms in WPI solutions and kept the samples sterilized for six months of shelf storage at room temperature. Control WPI solutions that did not received radiation treatment had 810 colonies forming units (cfu)/ml and 200 cfu/ml on total aerobic plate, and yeast & molds plate counts, respectively. Brookfield viscosity was used to measure the effects of gamma radiation on polymerization of the WPI solutions. The researchers observed that viscosity of 27% protein solution was increased from 2.19 for the control to 4.78 mPa.s for the sample treated at 25 KGy, respectively, and viscosity also increased during storage at 23°C. Overall, the significant increase in viscosity (mPa.s) of WPI solutions was most affected by the higher dosage of gamma radiation (15-25 KGy) and viscosity values also increased during the 6-month storage duration after treatment. Effects of gamma radiation and storage time on viscosity of whey protein solutions were significant (P<0.05). Also, turbidity (measurement of opacity) of WPI solutions increased from 0.14 to 0.16 for untreated and treated samples (35 KGy), respectively. Soluble nitrogen content was also used to measure the effects of gamma radiation on polymerization of WPI solutions. The team found that nitrogen solubility decreased significantly from 100-54.7% in WPI solution samples treated at 35 KGy due to protein-based conformational modifications. Radiation treatment had significant impact on turbidity and soluble nitrogen content of whey protein solutions (P<0.05). Polymerized interactions between whey proteins in solution were also analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Both 10 and 30% sample solutions showed that proteins were cross-linked in the samples treated by equal dosage of gamma radiation under the same amount of samples. Protein profiles suggested that non-quantitatively proteins were aggregated to higher molecular weights than samples untreated by gamma radiation. Also micrographs of TEM showed differences in protein-protein interactions induced by gamma radiation in treated WPI solutions, which displayed random and non-regular pattern protein structures. These affected structures seemed to be fragmented by gamma radiation and voids were created in the process. The researchers recommend additional research work to

1. Anderson, S. N., Richards, J. M., Esquer, H. J., Benninghoff, A. D., Arif, A. M., and Berreau, L. M. (2015). A Structurally-Tunable 3-Hydroxyflavone Motif for Visible Light-Induced Carbon Monoxide-Releasing Molecules (CORMs). ChemistryOpen 4, 590-594.
2. Andrade, J. E., Ju, Y. H., Baker, C., Doerge, D. R., and Helferich, W. G. (2015). Long-term exposure to dietary sources of genistein induces estrogen-independence in the human breast cancer (MCF-7) xenograft model. Mol Nutr Food Res 59, 413-423.
3. Andrade, J. E., Rosales, E., Lopez, J. R., Carrillo, E. P., Engeseth, N. J., and Helferich, W. G. (2015). Development of a point-of-use fortification technology for delivery of micronutrients in Honduras. J. Sci. Food Agric. 95, 393-400.
4. Andruska, N. D., Zheng, X., Yang, X., Mao, C., Cherian, M. M., Mahapatra, L., Helferich, W. G., and Shapiro, D. J. (2015). Estrogen receptor alpha inhibitor activates the unfolded protein response, blocks protein synthesis, and induces tumor regression. Proc. Natl. Acad. Sci. U. S. A. 112, 4737-4742.
5. Andruska, N., Zheng, X., Yang, X., Helferich, W. G., and Shapiro, D. J. (2015). Anticipatory estrogen activation of the unfolded protein response is linked to cell proliferation and poor survival in estrogen receptor alpha-positive breast cancer. Oncogene 34, 3760-3769.
6. Atwell, L.L., Beaver, L.M., Shannon, J., Williams, D. E., Dashwood, R.H. and Ho, E. (2015) Epigenetic Regulation by Sulforaphane: Opportunities for Breast and Prostate Cancer Chemoprevention. Current Pharmacol. Reports. 1:102-111.
7. Atwell, L.L., Hsu, A., Wong, C.P., Stevens, J.F., Bella, D., Yu, T.., Pereira, C.B., Löhr, C.V., Christensen, J.M., Dashwood, R.H., Williams, D.E., Shannon, J. and Ho, E. (2014) Absorption and Chemopreventive Targets of Sulforaphane in Humans Following Consumption of Broccoli Sprouts or Myrosinase-Treated Broccoli Sprout Extracts. Molec. Nutr. Food Res. 59:424-433.
8. Aumsuwan, P., Khan, S. I., Khan, I. A., Avula, B., Walker, L. A., Helferich, W. G., Katzenellenbogen, B. S., and Dasmahapatra, A. K. (2015). Evaluation of wild yam (Dioscorea villosa) root extract as a potential epigenetic agent in breast cancer cells. In Vitro Cell. Dev. Biol. Anim. 51, 59-71.
9. Bates, M.A., Brandenberger, C., Langohr, I., Kumagai, K., Harkema, J.R., Holian, A., Pestka, J.J., 2015. Silica Triggers inflammation and ectopic lymphoid neogenesis in the lungs in parallel with accelerated onset of systemic autoimmunity and glomerulonephritis in the lupus-prone NZBWF1 mouse. PLoS ONE 10, e0125481.
10. Bello NT 2015 Clinical utility of guanfacine extended release in the treatment of ADHD in children and adolescents. Patient Prefer Adherence 9:877-885
11. Bello NT, Walters AL, Verpeut JL, Caverly J 2014 Dietary-induced binge eating increases prefrontal cortex neural activation to restraint stress and increases binge food consumption following chronic guanfacine. Pharmacol Biochem Behav 125:21-28
12. Bello NT, Yeh CY, Verpeut JL, Walters AL 2014 Binge-like eating attenuates nisoxetine feeding suppression, stress activation, and brain norepinephrine activity. PLoS One 9:e93610
13. Benninghoff, A.D., Lefevre, M., Hintze, K.J., Ward, R.E. and Broadbent, J.R. (2015)  Fighting cancer with functional foods: new approaches to investigate the interactions of dietary bioactive chemicals and the gut microbiome.  Journal of Developments in Sustainable Agriculture 10: 1-21.
14. Bittner, G.D., Denison, M.S., Yang, C.S., Stoner, M.A. and He, G. (2014) Chemicals having estrogenic activity can be released from some bisphenol A-free, hard and clear, thermoplastic resins, Environ. Health 14 (1), 103.
15. Block, K. I., . . . . Helferich, W. G., et al. (2015). Designing a broad-spectrum integrative approach for cancer prevention and treatment. Semin. Cancer Biol. 35 Suppl, S276-304.
16. Boonmuen, N., Gong, P., Ali, Z., Chittiboyina, A. G., Khan, I., Doerge, D. R., Helferich, W. G., Carlson, K. E., Martin, T., Piyachaturawat, P., Katzenellenbogen, J. A., and Katzenellenbogen, B. S. (2015). Licorice root components in dietary supplements are selective estrogen receptor modulators with a spectrum of estrogenic and anti-estrogenic activities. Steroids.
17. Brennan, J. C., Bassal, A., He, G. and Denison, M.S. (2015) Development of a recombinant human ovarian (BG1) cell line containing estrogen receptor a and b for improved detection of estrogenic/antiestrogenic chemicals, Environ. Toxicol. Chem., In Press.
18. Brennan, J. C., He, G., Tsutsumi, T., Zhao, J., Wirth, E., Fulton, M. H. and Denison, M.S. (2015) Development of species-specific Ah receptor-responsive third generation CALUX cell lines with increased sensitivity and responsiveness, Environ. Sci. Tech. 49(19):11903-12.
19. Caron-Beaudoin, E., Denison, M. S. and Sanderson, J. T. (2015) Effects of neonicotinoids on promoter-specific expression and activity of aromatase in human adrenocortical carcinoma (H295R) and umbilical vein endothelial (HUVEC) cells, Toxicol. Sci., In Press.
20. Casey, S. C., Amedei, A., Aquilano, K., Azmi, A. S., Benencia, F., Bhakta, D., Bilsland, A. E., Boosani, C. S., Chen, S., Ciriolo, M. R., Crawford, S., Fujii, H., Georgakilas, A. G., Guha, G., Halicka, D., Helferich, W. G., Heneberg, P., Honoki, K., Keith, W. N., Kerkar, S. P., Mohammed, S. I., Niccolai, E., Nowsheen, S., Vasantha Rupasinghe, H. P., Samadi, A., Singh, N., Talib, W. H., Venkateswaran, V., Whelan, R. L., Yang, X., and Felsher, D. W. (2015). Cancer prevention and therapy through the modulation of the tumor microenvironment. Semin. Cancer Biol. 35 Suppl, S199-223.
21. Chieng, M., Ying, S., Kunde, D., Cook, A., Nerurkar, P.V., Ahuja, K. and Eri, R. Bitter melon extract protects against ER stress in LS174T colonic epithelial cells and copper-induced oxidation in human serum. Phytotherapy Research. Under revision, 2015
22. Chung, J., Anderson, S.A., Gwynn, B, Deck, K.M., Chen, M.J., Langer, N.B., Shaw, G.C., Huston, N.C., Boyer, L.F., Datta, S., Paradkar, P.N., Li, L., Wei, Z., Lambert, A.J., Sahr, K., Wittig, J.G., Chen, W., Lu, W., Galy, B., Schlaeger, T.M., Hentze, M.W., Ward, D.M., Kaplan, J., Eisenstein, R.S., Peters, L.L., Paw, B.H. (2014) Iron regulatory protein 1 protects against mitoferrin-I deficient porphyria. J. Biol. Chem. 289:7835. PMC3953295.
23. Chung, J., Bauer, D.E., Ghamari, A., Nizzi, C.P., Deck, K.M., Kingsley, P.D., Yuen, Y.Y., Huston, N.C., Chen, C., Schultz, I.J., Dalton, A.J., Wittig, J.G., Palis, J., Orkin, S.H., Lodish, H.F., Eisenstein, R.S., Cantor, A.B. and Paw, B.H. (2014) The mTORC/4E-BP pathway coordinately regulates hemoglobin production with L-leucine availability. Sci Signal. 2015 8(372):ra34. PMC4402725.
24. Coulombe, R.A., Bergeson, J., Aguilar, S., Hergert, N., and Lefevre, M. (2014) Effects of Ambient Particulate Air Pollution on Pulmonary Function, Pulmonary and Systemic Inflammation, and Lack Protective Effect of a Comprehensive Antioxidant and Anti-Inflammatory Dietary Supplement. PLOS One (in press).
25. Cox-York K, Sheflin AM, Foster MT, Gentile CL, Kahl A, Koch L, Britton S, Weir TL (2015) Gut ecology associated with high innate aerobic capacity protects against ovariectomy-induced cardiometabolic risk. Physiol Reports 3, e12488 .
26. Croes, K., Debaille, P., Van den Bril, B., Staelens, J., Vandermarken, T., Van Langenhove, K., Denison, M.S., Leermakers, M. and Elskens, M. (2015) Assessment of estrogenic activity in PM₁₀ air samples with the ERE-CALUX bioassay: method optimization and implementation at an urban location in Flanders (Belgium), Chemosphere 144, 392-398.
27. Davis, A. P., Benninghoff, A. D., Thomas, A. J., Sessions, B. R., and White, K. L. (2015). DNA methylation of the LIN28 pseudogene family. BMC Genomics 16, 287.
28. DeGroot, D.E., Franks, D.G., Higa, T., Tanaka, J., Hahn, M.E. and Denison, M.S. (2015) Naturally-occurring marine brominated indoles are aryl hydrocarbon receptor ligands/agonists, Chem. Res. Toxicol. 28, 1176-1185.
29. Ferguson, L. R., Chen, H., Collins, A. R., Connell, M., Damia, G., Dasgupta, S., Malhotra, M., Meeker, A. K., Amedei, A., Amin, A., Ashraf, S. S., Aquilano, K., Azmi, A. S., Bhakta, D., Bilsland, A., Boosani, C. S., Chen, S., Ciriolo, M. R., Fujii, H., Guha, G., Halicka, D., Helferich, W. G., Keith, W. N., Mohammed, S. I., Niccolai, E., Yang, X., Honoki, K., Parslow, V. R., Prakash, S., Rezazadeh, S., Shackelford, R. E., Sidransky, D., Tran, P. T., Yang, E. S., and Maxwell, C. A. (2015). Genomic instability in human cancer: Molecular insights and opportunities for therapeutic attack and prevention through diet and nutrition. Semin. Cancer Biol. 35 Suppl, S5-S24.
30. Gentile CL, Weir TL , Cox-York K , Yuren W, Wang D, Reese L, Moran G, Estrada A, Mulligan C, Pagliassotti M, Foster MT (2014) The Role of Visceral and Subcutaneous Adipose Tissue Fatty Acid Composition in Liver Pathophysiology Associated with NALFD. Adipocyte, doi=10.4161/21623945.2014.978662
31. Hintze, K.J, Cox, J.E., Rompato, G., Benninghoff, A.D., Ward, R.E., Broadbent, J., and Lefevre, M. (2014)  Broad scope method for creating humanized animal models for animal health and disease research through antibiotic treatment and human fecal transfer.  Gut Microbes 5(2): 1-9
32. Hong, M.Y., N.D. Turner, M.E. Murphy, R.J. Carroll, R.S. Chapkin, and J.R. Lupton.  In vivo regulation of colon cell proliferation, differentiation, apoptosis and P27Kip1 by dietary fish oil and butyrate in rats.  Cancer Prevention Research (In Press).
33. Jiang, W. G., Sanders, A. J., Katoh, M., Ungefroren, H., Gieseler, F., Prince, M., Thompson, S. K., Zollo, M., Spano, D., Dhawan, P., Sliva, D., Subbarayan, P. R., Sarkar, M., Honoki, K., Fujii, H., Georgakilas, A. G., Amedei, A., Niccolai, E., Amin, A., Ashraf, S. S., Ye, L., Helferich, W. G., Yang, X., Boosani, C. S., Guha, G., Ciriolo, M. R., Aquilano, K., Chen, S., Azmi, A. S., Keith, W. N., Bilsland, A., Bhakta, D., Halicka, D., Nowsheen, S., Pantano, F., and Santini, D. (2015). Tissue invasion and metastasis: Molecular, biological and clinical perspectives. Semin. Cancer Biol. 35 Suppl, S244-275.
34. Johnson, N.B., Deck, K.M., Nizzi, C.P. and Eisenstein, R.S. Interrelationships Between Iron-Sulfur Cluster Biogenesis and Control of Iron Metabolism by Iron Regulatory Protein 1 and the E3 Ubiquitin Ligase FBXL5. In preparation.
35. Kang, Y., Nian, H., Rajendran, P., Kim, E., Dashwood, W.M., Pinto, J., Boardman, L., Thibodeau, S., Limburg, P., Löhr, C., Bisson, W., Williams, D.E., Ho, E. and Dashwood, R.H. (2014) HDAC8 and STAT3 Repress BMF Gene Activity in Colon Cancer Cells. Cell Death Dis. 5:e1476.
36. Kelly CJ, Zheng L, Campbell EL, Saeedi B, Scholz CC, Bayless AJ, Wilson KE, Glover LE, Kominsky DJ, Magnuson AM, Weir TL, Ehrentraut SE, Nguyen V, Taylor CT, Colgan SP (2015) Host-Microbe Crosstalk between Short-Chain Fatty Acids and Intestinal Epithelial HIF Provides a New Mechanism to Augment Tissue Barrier Function. Cell Host & Microbe DOI: http://dx.doi.org/10.1016/j.chom.2015.03.005.
37. Khedidji, S., Croes, K., Yassaa, N., Ladji, R., Denison, M.S., Baeyens, W. and Elskens, M. (2015) Assessment of dioxin-like activity in PM₁₀ air samples from an industrial location in Algeria, using the DRE-CALUX bioassay, Environ. Sci. Poll. Res., In Press.
38. Kim, E., Bisson ,W.H., Löhr, C.V., Williams, D.E., Ho, E., Dashwood, R.H. and Rajendran, P. (2015) Histone and Non-Histone Targets of Dietary Deacethylase Inhibitors. Curr. Topics Med. Chem., in press.
39. Kim, E., L.A. Davidson, R.S. Zoh, B.S. Patil, G.K. Jayaprakasha, E.S. Callaway, C.D. Allred, N.D. Turner, and R.S. Chapkin.  Homeostatic responses of colonic LGR5 stem cells following acute in vivo exposure to a genotoxic carcinogen.  Carcinogenesis (Accepted with revision).
40. Liu, Y., Hilakivi-Clarke, L., Zhang, Y., Wang, X., Pan, Y. X., Xuan, J., Fleck, S. C., Doerge, D. R., and Helferich, W. G. (2015). Isoflavones in soy flour diet have different effects on whole-genome expression patterns than purified isoflavone mix in human MCF-7 breast tumors in ovariectomized athymic nude mice. Mol Nutr Food Res 59, 1419-1430.
41. Maayah, Z.H., Ghebeh, H.B., Alhaider, A.A., El-Kadi, A.O., Soshilov, A.A., Denison, M.S., Ansari, M.A. and Korashy, H.M. (2015) Metformin inhibits 2,7-dimethylbenz[a]anthracene-induced breast carcinogenesis and adduct formation in human breast cells by inhibiting the cytochrome P4501A1/aryl hydrocarbon receptor signaling pathway, Toxicol. Appl. Pharmacol. 284, 217-226. 
42. Madak-Erdogan, Z., Gong, P., Zhao, Y. C., Xu, L., Wrobel, K. U., Hartman, J. A., Wang, M., Cam, A., Iwaniec, U. T., Turner, R. T., Twaddle, N. C., Doerge, D. R., Khan, I. A., Katzenellenbogen, J. A., Katzenellenbogen, B. S., and Helferich, W. G. (2015). Dietary licorice root supplementation reduces diet-induced weight gain, lipid deposition, and hepatic steatosis in ovariectomized mice without stimulating reproductive tissues and mammary gland. Mol Nutr Food Res.
43. Madeen, E., Corley, R.A., Crowell, S., Turteltaub, K., Ognibene, T., Malafatti, M., McQuistan, T.J., Garrard, M., Sudakin, D. and Williams, D.E. (2014) Human In Vivo Pharmacokinetics of [¹⁴C]Dibenzo[def,p]chrysene by Accelerator Mass Spectrometry Following Oral
44. Mexia, N., Gaitanis, G., Velegraki, A., Soshilov, A., Denison, M.S. and Magiatis, P. (2015) Pityriazepin and other potent AhR ligands isolated from Malassezia furfur yeast, Arch. Biochem. Biophys. 571, 16-20.
45. Mohammad, R. M., Muqbil, I., Lowe, L., Yedjou, C., Hsu, H. Y., Lin, L. T., Siegelin, M. D., Fimognari, C., Kumar, N. B., Dou, Q. P., Yang, H., Samadi, A. K., Russo, G. L., Spagnuolo, C., Ray, S. K., Chakrabarti, M., Morre, J. D., Coley, H. M., Honoki, K., Fujii, H., Georgakilas, A. G., Amedei, A., Niccolai, E., Amin, A., Ashraf, S. S., Helferich, W. G., Yang, X., Boosani, C. S., Guha, G., Bhakta, D., Ciriolo, M. R., Aquilano, K., Chen, S., Mohammed, S. I., Keith, W. N., Bilsland, A., Halicka, D., Nowsheen, S., and Azmi, A. S. (2015). Broad targeting of resistance to apoptosis in cancer. Semin. Cancer Biol. 35 Suppl, S78-S103.
46. Monson, M. S., Coulombe, R.A., and K.M. Reed. (2015). Sensitivity and Response of Poultry to Aflatoxin B₁ Exposure. Agriculture   doi:10.3390/agriculture50x000x (in press).
47. Monson, M. S., Settlage, R.E., McMahon, K.W., Mendoza, K.M., Rawal, S., El-Nezami, H., Coulombe, R.A., and K.M. Reed. (2014) Response of the Hepatic Transcriptome to Aflatoxin B₁ in Domestic Turkey. PLOS One June 30;9(6): doi: 10.1371/journal.pone.0100930. 
48. Monson, M. S., Settlage, R.E., Mendoza, K.M., Rawal, S., El-Nezami, H., Coulombe, R.A., and K.M. Reed. (2015). Modulation of the Spleen Transcriptome in Domestic Turkey (Meleagris gallopavo) in Response to Aflatoxin B1 and Probiotics. Immunogenetics Jan 20; DOI 10.1007/s00251-014-0825-y
49. Neale, P., Ait-Aissa, S., Brack, W., Creusot, N., Denison, M.S., Deutschmann, B., Hilscherova, K., Hollert, H., Krauss, M., Novak, J., Schulze, T., Seiler, T.-B., Serra, H., Shao, Y. and Escher, B. (2015) Linking in vitro effects and detected organic micropollutants in surface water using mixture toxicity modeling, Environ. Sci. Tech., In Press.
50. Nerurkar PV, Hwang PW, Saksa E. Anti-Diabetic Potential of Noni: The Yin and the Yang. Molecules. 2015 Sep 25;20(10):17684-719. doi: 10.3390/molecules201017684. Review
51. Pan, X., Whitten, D.A., Wilkerson, C.G., Pestka, J.J., 2014. Dynamic changes in ribosome-associated proteome and phosphoproteome during deoxynivalenol-induced translation inhibition and ribotoxic stress. Toxicol Sci 138, 217-233.
52. Pestka, J.J., Vines, L.L., Bates, M.A., He, K., Langohr, I., 2014. Comparative effects of n-3, n-6 and n-9 unsaturated fatty acid-rich diet consumption on lupus nephritis, autoantibody production and CD4+ T cell-related gene responses in the autoimmune NZBWF1 mouse. PloS one 9, e100255.
53. Rajendran, P., Dashwood, W.-M., Li, L., Kang, Y., Kim, E., Johnson, G., Fischer, K.A., Löhr, C.V., Williams, D.E., Ho, E., Yamamoto, M., Lieberman, D.A. and Dashwood, R.H. (2015) Nrf2 Status Affects Tumor Growth, HDAC3 Gene Promoter Associations, and the Response to Sulforaphane in the Colon. Clin. Epigenetics 7:102.
54. Rawal, S., Bauer, M. M., Mendoza, K.M., El-Nezami, H., Hall, J.O., Kim, J.-E., Stevens, J.R., Reed, K.M., and R.A. Coulombe (2014). Aflatoxicosis Chemoprevention by Probiotic Lactobacillus and its Impact on BG Genes of the Major Histocompatibility Complex. Research in Veterinary Science. Jun 13. pii: S0034-5288(14)00170-2. doi: 10.1016/j.rvsc.2014.06.008.
55. Rensvold JW, Ong SE, Jeevananthan A, Carr SA, Mootha VK and Pagliarini DJ, Complementary RNA and Protein Profiling Identifies Iron as a Key Regulator of Mitochondrial Biogenesis. Cell Reports, 2013, 3(1): 237-245   Cover Article
56. Riley, R.T. (2014) Naturally Occurring Chemical Carcinogens. In: World Cancer Report 2014, pp. 169-176,  B. Stewart and C.P. Wild, eds., International Agency for Research on Cancer, Lyon, France.
57. Riley, R.T. and Miller, J.D. 2015. The Statement That Some Ochratoxins Are  “. . .classified as human carcinogens” Is Not Accurate. Toxicological Sciences 143:2
58. Riley, R.T., Showker, J.L., Lee, C.M., Zipperer, C.E., Mitchell, T.R., Voss, K.A., Zitomer, N.C., Torres, O., Matute, J., Gregory, S.G., Ashley-Koch, A.E., Maddox, J.R., Gardner, N., Gelineau-van Waes, J.B. 2015. A blood spot method for detecting fumonisin-induced changes in putative sphingolipid biomarkers in LM/Bc mice and humans. Food Additives and Contaminants, part A, 32(6): 934-949.
59. Riley, R.T., Torres, O., Matute, J.,  Gregory, S.G., Ashley-Koch, A.E.,  Showker, J.L., Mitchell, T.,  Voss, K.A.,  Maddox, J.R. and Gelineau-van Waes, J.B.  2015. Evidence for fumonisin inhibition of ceramide synthase in humans living in high exposure communities in Guatemala. Molecular Nutrition and Food Research (DOI: 10.1002/mnfr.201500499).
60. Ritchie, L.E., J.M. Sturino, R.J. Carroll, L.W. Rooney, A. Azcarate-Peril, and N.D. Turner.  2015a.  Polyphenol-rich sorghum brans alter colon microbiota and impacts species diversity and species richness after multiple bouts of dextran sodium sulfate-induced colitis. FEMS Microbiology Ecology 91: http://dx.doi.org/10.1093/femsec/fiv008.
61. Ritchie, L.E., S.S. Taddeo, B.R. Weeks, F. Lima, S.A. Bloomfield, M.A. Azcarate-Peril, S.R. Zwart, S.M. Smith, and N.D. Turner.  2015b.  Space environmental factor impacts upon murine colon microbiota and mucosal homeostasis. PLoS ONE DOI: 10.1371/journal.pone.0125792.
62. Samadi, A. K., Bilsland, A., Georgakilas, A. G., Amedei, A., Amin, A., Bishayee, A., Azmi, A. S., Lokeshwar, B. L., Grue, B., Panis, C., Boosani, C. S., Poudyal, D., Stafforini, D. M., Bhakta, D., Niccolai, E., Guha, G., Vasantha Rupasinghe, H. P., Fujii, H., Honoki, K., Mehta, K., Aquilano, K., Lowe, L., Hofseth, L. J., Ricciardiello, L., Ciriolo, M. R., Singh, N., Whelan, R. L., Chaturvedi, R., Ashraf, S. S., Shantha Kumara, H. M., Nowsheen, S., Mohammed, S. I., Keith, W. N., Helferich, W. G., and Yang, X. (2015). A multi-targeted approach to suppress tumor-promoting inflammation. Semin. Cancer Biol. 35 Suppl, S151-184.
63. Sheflin AM, Borresen EC, Wdowik MJ, Rao S, Brown RJ, Heuberger AL, Broeckling CD, Weir TL, Ryan EP (2015) Dietary intervention with heat-stabilized rice bran modulates gut microbiota and metabolites in healthy participants. Nutrients 7, 1282-1300.
64. Sheflin AM, Whitney AK, Weir TL (2014) Cancer Promoting Effects of Gut Microbial Dysbiosis. Current Oncology Reports 16, 406.
65. Siddens, L.K., Bunde, K.L., Harper, T.A. Jr., McQuistan, T.J., Löhr, C.V., Bramer, L.M., Waters, K.M., Tilton, S.C., Krueger, S.K., Williams, D.E. and Baird, W.M. (2015) Cytochrome P450 1b1 in Polycyclic Aromatic Hydrocarbon (PAH)-Induced Skin Carcinogenesis: Tumorigenicity of Individual PAHs and Coal-Tar Extract, DNA Adduction and Expression of Select Genes in the Cyp1b1 Knockout Mouse. Toxicol. Appl. Pharmacol. 287:149-160. PMCID in progress.
66. Strakovsky, R. S., Lezmi, S., Shkoda, I., Flaws, J. A., Helferich, W. G., and Pan, Y. X. (2015). In utero growth restriction and catch-up adipogenesis after developmental di (2-ethylhexyl) phthalate exposure cause glucose intolerance in adult male rats following a high-fat dietary challenge. J Nutr Biochem 26, 1208-1220.
67. Strakovsky, R. S., Wang, H., Engeseth, N. J., Flaws, J. A., Helferich, W. G., Pan, Y. X., and Lezmi, S. (2015). Developmental bisphenol A (BPA) exposure leads to sex-specific modification of hepatic gene expression and epigenome at birth that may exacerbate high-fat diet-induced hepatic steatosis. Toxicol. Appl. Pharmacol. 284, 101-112.
68. Tilton, S.C., Siddens, L.K., Krueger, S.K., Larkin, A.J., Löhr, C.V., Williams, D.E., Baird, W.M. and Waters, K.M. (2015). Mechanism-Based Classification of PAH mixtures to Predict Carcinogenic Potential. Toxicol. Sci. 146:135-145. PMCID: PMC4476464
69. Torres, O., Matute, J., Gelineau-van Waes, J., Maddox, J.R., Gregory, S.G., Ashley-Koch, A.E., Showker, J.L., Voss, K.A., Riley, R.T.  2015. Human health implications from co-exposure to aflatoxins and fumonisins in maize-based foods in the Latin America: Guatemala a case study. World Mycotoxin Journal 8 (2):143-159.
70. trichothecenes T-2 toxin, HT-2 toxin and satratoxin G to the ipecac alkaloid
71. Vandermarken, T., De Galan, S., Croes, K., Van Langenhove, K., Vercammen, J., Sanctorum, H., Denison, M.S., Goeyens, L., Elskens, M., and Baeyens, W. (2015) Characterisation and implementation of the ERE-CALUX bioassay on indoor dust samples of kindergartens to assess estrogenic potencies, J. Steroid Biochem. Molec. Biol. S0960-0760.
72. Vasta, J.D., Andersen, K.A., Deck, K.M., Nizzi, C.P., Eisenstein, R.S. and Raines, R.T. Selective inhibition of collagen prolyl-4-hydroxylase in human cells. ACS Chemical Biology (2015) in press.
73. Verpeut JL, Bello NT 2014 Drug safety evaluation of naltrexone/bupropion for the treatment of obesity. Expert Opin Drug Saf 13:831-841
74. Vinay, D. S., Ryan, E. P., Pawelec, G., Talib, W. H., Stagg, J., Elkord, E., Lichtor, T., Decker, W. K., Whelan, R. L., Kumara, H. M., Signori, E., Honoki, K., Georgakilas, A. G., Amin, A., Helferich, W. G., Boosani, C. S., Guha, G., Ciriolo, M. R., Chen, S., Mohammed, S. I., Azmi, A. S., Keith, W. N., Bilsland, A., Bhakta, D., Halicka, D., Fujii, H., Aquilano, K., Ashraf, S. S., Nowsheen, S., Yang, X., Choi, B. K., and Kwon, B. S. (2015). Immune evasion in cancer: Mechanistic basis and therapeutic strategies. Semin. Cancer Biol. 35 Suppl, S185-198.
75. Voreades N, Kozil A, Weir TL (2014) Diet and Development of the Intestinal Microbiome. Frontiers in Microbiology 5,494
76. Watson, G.W., Wickramasekara, S., Fang, Y., Maier, C.S., Williams, D.E., Dashwood, R.H., Perez, V.I. and Ho, E. (201x) HDAC Activity is not Required for Basal Autophagic Flux in Metastatic Prostate Cancer Cells. Exptl. Biol. Med., in press.
77. Watson, G.W., Wickramasekara, S., Fang, Y., Palomera-Sanchez, Z., Maier, C.S., Williams, D.E., Dashwood, R.H., Perez, V.I. and Ho, E. (2015). Analysis of Autophagic Flux in Response to Sulforaphane in Metastatic Prostate Cancer Cells. Submitted to Molec. Nutr. Fd. Res. June 24: doiL10.1002/mnfr.201500283. [epub ahead of print] PMID:26108801.
78. Watson, G.W., Wickramasekara, S., Maier, C.S., Williams, D.E., Dashwood, R.H. and Ho, E. (2015) Assessment of Global Proteome in LNCaP Cells by 2D-RP/RP LC-MS/MS Following Sulforaphane Exposure.  EuPA Open Proteomics Aug. 15, 2015, DOI 10.1016/j.euprot.2015.08.002.
79. Watson, G.W., Wickramasekara, S., Palomera-Sanchez, Z., Black, C., Maier, C.S., Williams, D.E., Dashwood, R.H. and Ho, E. (2014)  SUV39H1/H3K8me3 Attenuates Sulforaphane-Induced Apoptotic Signaling in PC3 Prostate Cancer Cells. Oncogenesis 3:e131. PMCID: PMC3787240.
80. Whitney AK and Weir TL (2015) Inhibitory activity of Fuzhuan tea fractions against Salmonella enterica and enteric pathogens. Current Topics in Phytochemistry (accepted).
81. Whitney AK and Weir TL (2015) Interaction of caffeine with the SOS response pathway in Escherichia coli. Gut Pathogens 7:21.
82. Wu W, Zhou HR, Pan X, Pestka JJ. 2015. Comparison of anorectic potencies of the
83. Wu, F., Groopman, J.D., Pestka, J.J., 2014. Public health impacts of foodborne mycotoxins. Annu Rev Food Sci Technol 5, 351-372.
84. Wu, W., He, K., Zhou, H.R., Berthiller, F., Adam, G., Sugita-Konishi, Y., Watanabe, M., Krantis, A., Durst, T., Zhang, H., Pestka, J.J., 2014. Effects of oral exposure to naturally-occurring and synthetic deoxynivalenol congeners on proinflammatory cytokine and chemokine mRNA expression in the mouse. Toxicol Appl Pharmacol 278, 107-115.
85. Wu, W., Zhou, H.R., Bursian, S.J., Link, J.E., Pestka, J.J., 2015. Emetic responses to T-2 toxin, HT-2 toxin and emetine correspond to plasma elevations of peptide YY and 5-hydroxytryptamine. Arch Toxicol.(in press, epub)
86. Wu, W., Zhou, H.R., Bursian, S.J., Pan, X., Link, J.E., Berthiller, F., Adam, G., Krantis, A., Durst, T., Pestka, J.J., 2014. Comparison of anorectic and emetic potencies of deoxynivalenol (vomitoxin) to the plant metabolite deoxynivalenol-3-glucoside and synthetic deoxynivalenol derivatives EN139528 and EN139544. Toxicol Sci 142, 167-181.
87. Wu, W., Zhou, H.R., He, K., Pan, X., Sugita-Konishi, Y., Watanabe, M., Zhang, H., Pestka, J.J., 2014. Role of cholecystokinin in anorexia induction following oral exposure to the 8-ketotrichothecenes deoxynivalenol, 15-acetyldeoxynivalenol, 3-acetyldeoxynivalenol, fusarenon X, and nivalenol. Toxicol Sci 138, 278-289.
88. Yang, X., Belosay, A., Hartman, J. A., Song, H., Zhang, Y., Wang, W., Doerge, D. R., and Helferich, W. G. (2015). Dietary soy isoflavones increase metastasis to lungs in an experimental model of breast cancer with bone micro-tumors. Clin. Exp. Metastasis 32, 323-333.
89. Zhao, N., Nizzi, C.P., Anderson, S.A., Wang, J., Ueno, A., Tsukamoto, H., Eisenstein, R.S., Enns, C.A. and Zhang, A-S. (2014) Low intracellular iron increases the stability of matriptase-2. J. Biol. Chem. 290:4432-46. PMC4326848
90. Zhou, H.R., He, K., Landgraf, J., Pan, X., Pestka, J.J., 2014. Direct activation of ribosome-associated double-stranded RNA-dependent protein kinase (PKR) by deoxynivalenol, anisomycin and ricin: a new model for ribotoxic stress response induction. Toxins (Basel) 6, 3406-3425.
91. Zhou, H.R., Pestka, J.J., 2015. Deoxynivalenol (Vomitoxin)-induced cholecystokinin and glucagon-like peptide-1 release in the STC-1 enteroendocrine cell model is mediated by calcium-sensing receptor and transient receptor potential ankyrin-1 channel. Toxicol Sci 145, 407-417.