bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolism of cysteine in primary hepatocytes from cysteine dioxygenase (CDO1) knockout mice
Authors: Jurkowska Halina , Hirschberger Lawrence L. , Roman Heather B , Wróbel Maria , Stipanuk Martha H.
Published: June 2021

Cysteine dioxygenase (CDO1) knockout mice have higher tissue cysteine levels and metabolize cysteine to H2S by alternative desulfhydration pathways catalyzed by cystathionine β‐synthase (CBS) and cystathionine γ‐lyase (CTH), and mercaptopyruvate sulfurtransferase (MPST). We evaluated the effects of a lack of CDO1 in hepatocytes isolated from wild‐type (CDO+/+) or CDO null (CDO−/−) mice. CDO−/− hepatocytes had intracellular cysteine levels that were 1.8 to 2.8‐times the levels in CDO+/+ cells, and similarly elevated glutathione levels. Increasing the cyst(e)ine concentration of the culture medium from 0.2 mM up to 0.7 mM increased intracellular cysteine and glutathione levels only slightly. Thus, both CDO+/+ and CDO−/− cells were able to regulate intracellular cysteine levels, although CDO−/− cells regulated cysteine levels at a higher steady‐state concentration. Cystathionine levels in CDO−/− cells were approximately double those in CDO+/+ cells, consistent with increased metabolism of cysteine (+homocysteine) to cystathionine + H2S by CBS. CTH activity, but not abundance, was about 50% higher in CDO−/− cells, consistent with possible upregulation of CTH in CDO−/− cells. The activity of MPST was not significantly increased in CDO−/− cells. Results are similar to those observed in liver of intact CDO−/− mice. Supported by NIH DK056649 and by a “Mobility Plus” fellowship from MNISW (Poland).
Potential roles for vitamins E and C in combination in modulation against exhaustive swimming‐and high altitude‐associated lung injury in rats
Authors: AL‐Hashem Fahaid , Alkhateeb Mahmoud
Published: June 2021

The aim is to evaluate and compare the potential role of vitamins E and C in protecting against acute swimming induced lung damage at different altitudes. Rats at both altitude were divided into nonstress control group, forced swimming stressed group and vitamin E and C pretreated stressed group. At the end of the procedure, lung tissue level of thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) were determined. In control rats, the baseline levels of TBARS were significantly increased and the baseline levels of both GSH and SOD were significantly decreased in the lungs of animals at high altitude than those at low altitude. Acute forced swimming resulted a significant increase in TBARS levels and a significant decrease in activities of SOD and CAT in the lungs in both altitude areas, and resulted in a significant decrease in GSH levels at high altitude rats only as compared with the resting state. Supplementation of vitamins E and C in combination effectively ameliorated all the parameters measured at both altitudes. Our novel observations suggest that supplementation of vitamins E and C could be beneficial against exhaustive swimming‐and high altitude‐associated lung injury.
Electrolyte‐free milk protein solution influences sodium and fluid retention in rats
Authors: Ishihara Kengo , Kato Yoshiho
Published: June 2021

Milk is an effective post‐exercise rehydration drink that maintains the net positive fluid balance. However, it is unclear which components are responsible for this effect. We assessed the effect of milk protein solution (MPS) obtained by dialysis on body fluid retention. Milk, MPS, MES (milk electrolyte solution), sports drink, and water were administered to male Wistar rats at a dose of 6 mL/rat after treadmill exercise. Total body fluid retention was assessed by urine volume 4 h after administration of hydrating liquids. Plasma osmolality, sodium and potassium levels, and urinary sodium and potassium were measured by HPLC and osmometry, respectively. During 4 h of rehydration, cumulative urine volumes differed significantly between treatment groups (p < 0.05) with 4.9, 2.2, and 3.4 mL from water‐, milk‐, and MPS‐fed rats, respectively. Thus, MPS elicited 50% of total body fluid retention of milk. Plasma aldosterone levels were significantly higher in MPS‐and milk‐fed rats compared to water‐fed rats. Plasma osmolality was maintained at higher levels in MPS‐fed rats than in water and MES‐fed rats (p < 0.05). Cumulative urine sodium excretion was also suppressed in the milk‐and MPS‐fed groups compared to the MES‐fed group. Our results demonstrate that MPS obtained by dialysis clearly affects net body water balance. This effect was attributed to retention of sodium and water, and maintenance of plasma osmolality.
Serum 25(OH)D does not correlate with BMI or cardiovascular fitness in young adults with sufficient vitamin D status
Authors: Scholten Shane D. , Overweg Taylor A. , Schenavar Brian M. , Sergeev Igor N.
Published: June 2021

Vitamin D supplementation is an emerging approach for enhancement of physical performance. Correlations have been found between low vitamin D status (circulating concentration of 25(OH)D) and cardiorespiratory fitness (CRF), however the evidence supporting the ergogenic effect of vitamin D in subjects with sufficient status of this vitamin is lacking. The purpose of this study was to investigate the relationship between serum 25(OH)D, CRF and body mass index (BMI) in young adults. Twenty physically active (4.1±3.4 h/wk, VO2peak=48.9±6.9 mL·kg−1·min−1), normal weight (BMI=22.9±2.2), college age (21.5±2.1) men and women participated in the study. Serum samples were analyzed for 25(OH)D using chemiluminescent EIA (Diazyme 25‐OH Vitamin D Assay). A two‐way ANOVA with interactions was used to compare differences between variables. The results showed that serum 25(OH)D concentration (25.7±6.0 ng/mL) had no correlation with VO2peak (p=0.510) or BMI (p=0.889) in men or women. All but one of the subjects had a 25(OH)D concentration >;20 ng/mL, and 5 subjects had 25(OH)D level >;30 ng/mL. The findings obtained indicated that serum levels of 25(OH)D, which are generally considered as sufficient, were not correlated with BMI or CRF in healthy, college‐aged men and women. Supported by NIH grant 8P20GM103443–12.
Vitamin D, calcium, potassium and calorie intakes associated with distinct breakfast patterns in adults (NHANES 2001–2008)
Authors: Fulgoni Victor L , Quann Erin E , Auestad Nancy
Published: June 2021

Foods consumed together at breakfast form distinct breakfast patterns. Cluster analyses using NHANES (2001–2008) data for adults (19+ y) identified 12 unique breakfast patterns. Two of the 12 patterns consisted predominantly of ready‐to‐eat cereal (RTEC) with milk (18% of adults). The other patterns were predominantly grain‐based, egg‐based, meat‐based, cooked cereal, beverage‐based, fruit, or no breakfast (38%, 7%, 3%, 5%, 9%, 2% and 19% of adults, respectively). Higher total daily consumption of dairy occurred with the 2 RTEC‐with‐milk patterns (2.1–2.4 cup‐eq.) vs. the other patterns including no breakfast (1.1–1.7 cup‐eq.); similarly for milk (1.4–1.7 vs. 0.4–0.9 cup‐eq., respectively). Total daily vitamin D intake was 6.7–7.3 vs. 2.3–5.1 mcg, respectively; calcium was 1096–1195 vs. 695–1009 mg, respectively; and potassium was 3009–3059 vs. 2013–3050 mg, respectively. Daily calories were 2132–2291 vs. 1967–2354 kcal, respectively. Breakfast patterns with higher milk intakes were associated with higher total daily vitamin D (3.6 mcg/cup‐eq.), calcium (381 mg/cup‐eq.) and potassium (553 mg/cup eq.). Little cheese (0–0.20 cup‐eq.) and yogurt (0–0.04 cup‐eq.) was consumed across all breakfast patterns. Total daily intakes of vitamin D, calcium and potassium, three of the nutrients of concern for Americans, are higher when milk is part of the breakfast meal. Supported by the Dairy Research Institute/National Dairy Council.
AIN‐76 and AIN‐93 diets do not provide adequate iron for perinatal development
Authors: Prohaska Joseph , Broderius Margaret
Published: June 2021

Iron supplementation during pregnancy and neonatal development remains controversial. Rodent models intending to evaluate trace element deficiencies and interactions should rely on an optimal control diet. When the AIN‐93 formulation was developed it dealt primarily with changes in carbohydrate, lipid, and calcium levels; iron recommendations remained the same as its predecessor diet, AIN‐76A, at 35 mg/kg. Increasing the dietary iron to 75 mg/kg (a level recommended by NRC guidelines) to pregnant Sprague Dawley rats, or postweanling males demonstrated that iron status is improved in dams, pups, as well as rapidly growing males. This is based on measurements of hepatic iron, plasma iron, and hemoglobin concentration. Phenotype of copper‐deficient (CuD) dams and pups fed modified AIN‐76A diets were different than CuD rats fed modified AIN‐93G diets with the higher iron level compared to copper‐adequate (CuA) controls. Hepatic iron was no longer augmented in the CuD groups. Additionally plasma and brain iron differences were attenuated. For example, brain iron deficiency evaluated by augmented transferrin receptor expression in CuD rats was not observed in the CuD pups reared by CuD dams fed the modified AIN‐93G diets with extra iron. It may be prudent to reevaluate the composition of standard “control” diets used in rodent models.

Supported by International Copper Association.
25‐HYDROXY D AND ITS RELATIONSHIP TO POTENTIAL SOURCES
Authors: Clausen Mary R. , Darr Rachel L. , Williams Brian T. , Millen Amy E. , Burton Harold W. , Browne Richard W. , Horvath Peter J.
Published: June 2021

Vitamin D status is associated with bone health and with cardiovascular health, diabetes mellitus and cancer. Since vitamin D status is affected by food and supplement intake and by sun exposure, the ability to accurately predict vitamin D status assessed from these sources is important. The objective of this study is to evaluate the use of 3‐day diet records (3DR) including dietary supplements and a food frequency questionnaire (FFQ) not including dietary supplements to predict serum 25‐hydroxyvitamin D concentrations [25(OH)D] in 40 male and female subjects (30 to 69 yrs). Time spent in sunlight was self‐reported along with a Fitzpatrick Skin Type Questionnaire. Vitamin D intake from the 3DR was positively correlated with 25(OH)D with a curvilinear relationship and may be used to predict 25(OH)D (rs=0.60, P <0.001). FFQ was not correlated with 25(OH)D (rs=0.15, P=0.38) but the FFQ with supplements was positively correlated (rs=0.42, P=0.013). Unprotected sun exposure or skin type did not aid the ability to predict 25(OH)D levels possibly due to winter month data collection with minimum unprotected sun exposure. Intake of vitamin D from supplements must be considered when estimating vitamin D status.
The expression of Apoc3 mRNA is regulated by HNF4α and COUP‐TFII levels, but not retinoid treatments, in rat primary hepatocytes and hepatoma cells
Authors: Howell Meredith , Li Rui , Zhang Rui , Chen Guoxun
Published: June 2021

Retinoic acid (RA) treatment induces hyperlipidemia in humans and animals. This has been attributed to the induction of Apolipoprotein CIII (gene, Apoc3), which inhibits lipoprotein lipase activity. We have shown that vitamin A status and retinoid treatment regulate hepatic lipogenic gene expression, suggesting that the induction of lipogenic genes, but not Apoc3, may contribute to hyperlipidemia. To test this, we analyzed the expression levels of Apoc3 mRNA in response to retinoid treatments or adenovirus‐mediated over‐expression of nuclear receptors mediating RA responses in primary rat hepatocytes and HL1C rat hepatoma cells using real‐time PCR. We report that retinoids did not induce Apoc3 mRNA expression in these cells. The over‐expression of HNF4α or COUP‐TFII significantly induced or inhibited the Apoc3 mRNA level, respectively, whereas RXRα or RARα over‐expression had no effect. We conclude that RA treatment could not directly induce Apoc3 mRNA levels in rat hepatocytes. Instead, the hepatic expression of Apoc3 mRNA is regulated by the expression levels of HNF4α and COUP‐TFII. Therefore, RA‐induced hyperlipidemia may not be attributed to the direct induction of Apoc3 mRNA level in hepatocytes.

Sponsor: Dr. Guoxun Chen, 1215 W. Cumberland Ave. 229 Jessie Harris Building, Knoxville, TN, 37996
Oral Administration of Retinoic Acid Lowers Brain Serotonin Concentration in Rats
Authors: Smazal Anne Lorraine , Lee Schalinske Kevin
Published: June 2021

Major depressive disorder has been associated with lowered functionality of serotonin, dopamine, and norepinephrine in the brain. Oral retinoid therapy has been reported to induce sudden and severe depressive symptoms in adolescents and adults. Retinoid compounds have been shown to negatively impact neurotransmitter synthesis and transport in vitro, whereas animal models of retinoid therapy and brain health have traditionally utilized intraperitoneal administration of retinoic compounds and focused on depressive behaviors. To our knowledge, no previous in vivo studies have used oral administration to demonstrate the impact of retinoid compounds on neurotransmitters in the brain. In this study, male Sprague‐Dawley rats received oral administration of either all‐trans‐retinoic acid or vehicle daily. After 9 days, rats were sacrificed and brain tissue was collected for analysis. Rats receiving all‐trans‐retinoic acid had a significant (48%) decrease in brain serotonin concentrations compared to control. Neither norepinephrine nor dopamine levels were impacted by oral dosage of all‐trans‐retinoic acid. To our knowledge, this is the first demonstration of lowered brain serotonin concentrations resulting from oral administration of retinoic acid and represents a potential therapeutic strategy to prevent major depressive disorder owing to retinoid usage. Support: College of Human Sciences, ISU.

Grant Funding Source: College of Human Sciences, ISU
The effect of curcumin plus piperine on body weight and fat loss as well as on the plasma levels of inflammatory cytokines in obese mice
Authors: Noble Timothy , Zingg Jean‐Marc , Paul Ligi , Smith Donald , Meydani Mohsen
Published: June 2021

Obesity is a major risk factor for CVD, diabetes, and other chronic diseases. Identifying dietary component(s) that can accelerate BW loss in overweight and obese individuals undergoing a BW reduction regimen will be of great value. Current evidence supports the concept that inclusion of curcumin (Cur) in a food restricted (FR) diet would facilitate BW loss in overweight individuals. To test this hypothesis, we fed 47 mice a high fat (HF) diet for 6 mo to increase their BW. The mice were then divided into 5 groups. One group continued on HF diet ad‐libitum. Four groups were FR by 20% with or without Cur (1000mg/kg diet) and piperine (Pip) (50mg/kg diet) for an additional 8 mo. BWs were reduced due to body fat loss measured by MRI. Cur alone or combined with Pip in FR mice had no additional effects on total BW and fat loss. FR alone reduced plasma levels of IL‐1 and KC/GRO, but FR groups supplemented with Cur+Pip further reduced plasma levels of several inflammatory cytokines including IL‐1, IL‐6, IL‐10 and KC/GRO. While FR‐primed peritoneal macrophages showed lower lipids uptake, Cur and Pip supplementation increased lipid accumulation in these cells. Of note, FR reduced telomere length in liver cells whereas Cur+Pip prevented the FR effect. These results suggest that a more effective health impact is achievable when Cur is administered together with Pip. NIFA/USDA grant #2010–65200‐20395 &USDA contract #58–1950‐0–014.
The synergistic effect of 1′‐acetoxychavicol acetate and sodium butyrate on cell death of human hepatocellular carcinoma cells
Authors: Kato Rie , Matsui‐Yuasa Isao , Azuma Hideki , Kojima‐Yuasa Akiko
Published: June 2021

Introduction

1′‐Acetoxychavicol acetate (ACA), obtained from alpinia galangal, exhibits chemopreventive effects on chemically induced tumor formation. Sodium butyrate (NaB), one of the major short chain fatty acids, is known to inhibit cancer cell proliferation in tumor cells. The interaction between ACA and NaB is still unclear. Here, we examined the effect of ACA and NaB on cell death of HepG2 cells.

Methods

The cell number was measured by neutral red assay. Intercellular reactive oxygen species (ROS) was measured by DCFH‐DA assay.

Results

The cell number of HepG2 cells was synergistically decreased with the combined treatment of ACA and NaB, resulting from apoptotic cell death. In ACA and NaB simultaneously treated cells, intercellular ROS also increased significantly. However, the cell number in ACA and NaB treated cells was improved with the treatment of catalase. The increase in intercellular ROS was prevented with DPI, the inhibitor of NADPH oxidase. These events weren't observed in normal cells.

Conclusion

ACA and NaB synergistically induce apoptotic cell death via the activation of NADPH oxidase and the increase in the intercellular ROS, and the effect is specific to carcinoma cells.
Genistein via JAK2 increased the phosphorylation of AMPK independently leptin receptor in C2C12 cells
Authors: Palacios‐Gonzalez Berenice , Flores‐Galicia Isabel , Torres Nimbe , Tovar Armando R
Published: June 2021

Consumption of soy protein in Zucker fa/fa rats increase the expression of genes involved in fatty acid oxidation as well as the phosphorylation of AMPK and ACC in skeletal muscle. Previous studies have shown that incubation of C2C12 with genistein, induces the expression of genes involved in fatty acid oxidation and phosphorylation of AMPK. The aim of the study was to determine whether genistein can increase the gene expression and phosphorylation of AMPK in cells silenced for the leptin receptor (ObR). Regardless of the leptin receptor, genistein increased the phosphorylation of AMPK and the expression of CPT1, UCP3, PCG1α, PPARδ. The AMPK phosphorylation induced by genistein was partially repressed when cells were incubated with the inhibitor of the kinase activity of JAK2. Interestingly in Zucker fa/fa rats which have a mutation of the leptin receptor and that consumed soy protein showed also an increased phosphorylation of JAK2 in skeletal muscle. These results indicate that genistein stimulates JAK2 phosphorylation, which in turn stimulates AMPK phosphorylation and expression of genes involved in fatty acid oxidation.

Grant Funding Source: CONACYT
Gene expression of hepatic cortisol binding globulin and enzymes modifying sex hormones are modulated in mice by feeding the nonfermentable viscous soluble dietary fiber, HPMC
Authors: Kim Hyunsook , Bartley Glenn E , Yokoyama Wallace
Published: June 2021

Global changes in hepatic gene expression in mice fed high fat diets and the nonfermentable viscous soluble dietary fiber, hydroxypropyl methylcellulose (HPMC) were determined by exon microarray. Obese C57BL/6J mice were fed either 6 % HPMC or 6 % microcrystalline cellulose (MCC) for five weeks. In addition to lowered plasma and liver lipids, body weight gain and adipose tissue weight were significantly lower in HPMC fed mice compared with MCC. We found that gene expression of CBG, the serpina6 gene, is decreased and that other sex hormone related genes were modulated by HPMC feeding. HPMC feeding also upregulated expression of genes related to PUFAs elongation. Cortisol, a steroid hormone, is regulated by the hypothalamic‐pituitary‐ adrenal (HPA). HPA affects obesity‐related chronic stress, which is also regulated differentially by sex hormones (such as androgens). Enzymes related to polyunsaturated fatty acid (PUFA) elongation have been shown to protect age‐related cognitive decline (ARCD). Therefore, in addition to already recognized positive effects on cholesterol and lipid metabolism HPMC intake may also affect neural functions such as gender‐related stress response and ARCD.
Variations in Milk Carotenoids from Jersey Cows fed Pasture versus Total Mixed Ration
Authors: Beliveau Amy Rose , Smith Jillian M , Wenzel Adam James , Curran‐Celentano Joanne
Published: June 2021

Interest in organic dairy production is rising. According to the USDA Certified Organic standards, producers are required to feed their cows on pasture for a minimum of 120 days. Research indicates that factors such as diet have the potential to impact milk composition. The objective of this experiment was to determine the changes in concentrations of milk carotenoids over a pasture season from cows under two different feeding systems. Individual milk samples were collected biweekly from 9 cows fed on pasture at an organic dairy and from 9 cows fed total mixed ration (TMR) at a conventional dairy. Components were extracted from the milk and separated via High Performance Liquid Chromatography. Data were analyzed using SPSS® version 19.0. Results showed that total carotenes, total xanthophylls, lutein, β‐cryptoxanthin, α‐ and β‐carotene and 13cis β‐carotene were significantly higher in milk from cows fed on pasture. These components also varied significantly over the season. In milk from cows fed TMR, only total xanthophylls and lutein changed significantly over time. Results indicate that pasture feeding affects carotenoid concentration in milk, suggesting that both diet and season can influence milk composition. Potential variations should be reflected in the National Nutrient Database.

Grant Funding Source: NH Agriculture Experiment Station
Ontogenic expression of retinoid homeostatic genes in principal vitamin A storage organs
Authors: Owusu Sarah A , Ross Catharine
Published: June 2021

Vitamin A (VA) is essential for proper lung development in early life. The combination of VA and 10% all‐trans retinoic acid (VARA) works synergistically to increase total retinyl ester storage and retinol uptake in neonatal rat lung. To confirm the necessity of VA for lung maturation and septation, VA metabolism during neonatal development must be understood. In the present study, we determined the effects of VA nutritional status and VARA supplementation during ontogeny on expression of retinoid homeostatic genes. Rat dams and their post‐weaning offspring were fed a VA‐marginal or supplemented diet, while pups received an oral dose of oil (placebo) or VARA on post‐natal day (P) P0/1, P4, P7, and P10. Tissues were collected 6 hr later. Gene expression was determined by qRT‐PCR in liver, lung, and kidney. VA‐supplemented diet and VARA treatment upregulated LRAT, Cyp26A1/B1, and STRA6, retinoid homeostatic genes important for VA storage, RA terminal oxidation, and retinol uptake, respectively. However, VARA did not regulate VA trafficking genes (RBP in liver and megalin in kidney). Cyp26a1 expression in liver decreased throughout development in rats fed marginal diet, but increased on the supplemented diet. No apparent difference was observed in RALDH1/2/3 expression with VARA treatment. Our results advance the understanding of neonatal VA metabolism and catabolism in principal VA storage organs.

Grant Funding Source: NIH HD006892
1,25‐dihydroxyvitamin D regulation of pyruvate carboxylase and glucose addiction in MCF10A‐ras human breast epithelial cells
Authors: Zheng Wei , Teegarden Dorothy
Published: June 2021

The mechanism and biological consequence of 1,25‐dihydroxyvitamin D (1,25D) regulation of glucose metabolism in progression to cancer (Warburg effect) was investigated. Our previous studies show that 1,25D shifts glucose metabolism towards reduced glycolysis and lactate production and reduced flux through the TCA cycle in ras‐oncogene transfected MCF10A (ras) cells, a model of early breast cancer progression. In the current studies, 1,25D reduced the mRNA (by 55%) and protein (by 35%) expression of pyruvate carboxylase (PC), a key enzyme for the anaplerosis of the TCA cycle, suggesting 1,25D may regulate glucose metabolism via PC. 1,25D inhibited growth, reducing ras cell number by 44% in media containing 5 mM glucose. Glucose restriction to 1 mM further reduced ras cell number by 39% compared to 5 mM glucose, indicating the ras cell dependence on glucose (addiction). However, 1,25D prevented the reduction in cell number at 1 mM glucose, suggesting 1,25D may reduce ras cell glucose addiction. Consistent with these results, flow cytometry analysis showed 1,25D treatment prevented the increase in G1 cell cycle arrest at 1 mM glucose noted in ras cells, further supporting that 1,25D reduces glucose addiction. These results suggest 1,25D regulation of glucose metabolism during early cancer progression has important biological consequences which may be mediated via PC.

Grant Funding Source: NIH R25CA128770
Control of metabolic genes in human mammary epithelial cells by vitamin D hinders adaptation to glutamine deprivation
Authors: Beaudin Sarah , Welsh JoEllen
Published: June 2021

The metabolic switch associated with cancer progression increases cellular requirements for nutrients, including the amino acid glutamine. The p53 tumor suppressor has been shown to abrogate the metabolic switch via effects on glutamine metabolism. Through microarray profiling of human mammary epithelial cells (HMEC) treated with 1,25‐dihydroxyvitamin D (1,25D) we discovered that the vitamin D receptor also targets genes involved in glutamine metabolism. qPCR assays in immortalized HMECs confirmed that 1,25D increased (>;10‐fold) the glutamate transporter SLC1A1 and reduced (>;4‐fold) the glutamine synthetase GLUL. Western blotting demonstrated decreased expression of glutamine synthetase (GS) and increased expression of SLC1A1 following 1,25D treatment. Because GS is essential for proliferation during glutamine depletion, we hypothesized that its inhibition by 1,25D would interfere with adaptation to low glutamine. Indeed, HMECs pretreated with 1,25D failed to adapt to glutamine deprivation, and high extracellular glutamine partially rescued 1,25D‐mediated growth arrest. Similar 1,25D‐mediated changes in GLUL and SLC1A1 were observed in pre‐neoplastic HMECs expressing SV40, but not in tumorigenic HMECs expressing SV40 and oncogenic ras. Collectively, these data implicate VDR in regulation of a critical node of the cancer‐associated metabolic switch. Grant number: R21CA166434
Apoptosis Induction in Hep3B cell line by Lu‐Do‐Huang
Authors: Yang Jo‐Hsuan , Chien Ting‐Yi , Tsai Chun‐Han , Huang Hui‐Yu
Published: June 2021

Pracparatum mungo (Lu‐Do‐Huang) is a derivative of Phaseolus mungo beans of the Leguminosae plant as a traditional medicine for liver detoxifying and anti‐liver cancer effect in southern China. The present study aimed to evaluate Lu‐Do‐Huang ethanol extract (LDHE) on anti‐liver cancer effect with Hep3B cell. The apoptotic activities of LDHE were assessed using flow cytometry, western blotting and immunohistochemical analysis. Our result showed that LDHE significantly inhibited the Hep3B cell growth. LDHE induced Hep3B cells to undergo apoptosis which was determined from Hep3B cell morphology change, increase of apoptotic bodies, apoptotic cells, DNA fragmentations and caspase activity. In conclusion, LDHE lead Hep3B cell to the apoptosis that can be used as precusors in development of anti‐liver cancer drugs. Suppression of apoptosis might contribute to tumor development by mean of accumulation of continuously proliferating cells.
α‐linolenic acid reduces growth in four breast cancer cell lines with varying receptor expression in high and low estrogen environments
Authors: Wiggins Ashleigh K.A. , Kharotia Shikhil , Mason Julie K. , Thompson Lilian U.
Published: June 2021

Breast cancer differs in cell receptors which then determines effective treatments. Alpha‐linolenic acid (ALA)‐rich flaxseed oil reduced estrogen receptor (ER)+ cell growth at low and high estrogen (E2) but its effect is unclear in cells with varying ER, progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). Our objective was to determine in vitro the ALA and E2 effect on growth and membrane lipids of four breast cancer cell lines with varying ER, PR and HER2 status. Cells were treated with 0 – 200uM ALA ± 1nM E2 and tested for growth by trypan blue exclusion method and fatty acids by gas chromatography. Receptor status confirmed by Western Blot: MCF7‐ER+PR+low HER2; BT474‐ER+PR+HER2+, MDA MB 231‐ER‐PR‐low HER2; MDA MB 468‐ER‐PR‐HER2‐. Compared to –E2 control, MCF7 had a significant E2, ALA and interaction effect on growth; other cell lines only had an ALA effect. 50uM and 75uM ALA significantly decreased growth by 14, 48, 31 and 63% and 38, 77, 80 and 78% in MCF7, BT 474, MDA MB231 and MDA MB468, respectively. E2 significantly increased (91.8%) MCF7 growth. Cells showed significant increases (9–25 fold) in % ALA which related to growth. Thus ALA reduces breast cancer cell growth ± E2 at doses as low as 50uM, likely through alteration of lipid composition. It is effective regardless of ER, PR and HER2 status which may be of importance in difficult to treat cancers such as triple negative. Funded by NSERC.

Grant Funding Source: NSERC
Conjugated Linoleic Acid Reduces Adiposity and Increases Markers of Browning and Inflammation in White Adipose Tissue of Mice
Authors: Shen Wan , Chuang Chia‐Chi , Martinez Kristina , Reid Tanya , Brown J. Mark , Xi Lin , Hixson Lindsay , Starnes Joe , McIntosh Michael
Published: June 2021

The objective of this study was to examine the mechanism by which conjugated linoleic acid (CLA) reduces body fat. Mice were fed three combinations of fatty acids at three doses (0.06%, 0.2%, and 0.6%, w/w) for 7 wk. The types of fatty acids were linoleic acid (control), an equal mixture of 10,12 CLA plus linoleic acid, or an equal mixture of 10,12 plus 9,11 CLA. Mice receiving the 0.2% and 0.6% dose of 10,12 CLA or the CLA mixture had decreased white adipose tissue (WAT) and brown adipose tissue (BAT) mass and increased incorporation of CLA isomers in epididymal WAT, liver, and muscle compared to controls. Food intake, glucose tolerance, and liver triglycerides were not affected by 0.2% CLA. Notably, in mice receiving 0.2% of both CLA treatments, the mRNA levels of genes associated with browning including uncoupling protein1 (UCP1), UCP1 protein levels, cytochrome c oxidase (CCO) activity, and the appearance of multilocular lipid droplets were most robustly increased in epididymal WAT compared to controls. CLA‐induced browning was accompanied by increases in mRNA levels of markers of low‐grade inflammation. Muscle CCO activity and BAT UCP1 protein levels were not affected by either CLA treatment. Collectively, these data suggest a linkage between decreased adiposity, browning in WAT, and low‐grade inflammation due to consumption of 10,12 CLA. Supported by NIH NIDDK/ODS 5R01‐DK063070‐09.

Grant Funding Source: NIH NIDDK/ODS 5R01‐DK063070‐09
Effects of Cfh genotype and dietary glycemic index on age‐related macular degeneration in mice
Authors: Rowan Sheldon , Weikel Karen , Chang Min‐Lee , Nagel Barbara , Thinschmidt Jeffrey , Grant Maria , Fliesler Steven , Taylor Allen
Published: June 2021

Age‐related macular degeneration (AMD) is a leading cause of visual impairment in aging Americans. Both genetics and diet contribute to the risk for developing AMD. For example, common variants in the Complement Factor H (Cfh) gene confer major susceptibility to‐ or protection from‐ developing AMD. Similarly, consuming diets with higher glycemic index (GI) foods is associated with increased risk of AMD. Understanding genetic/diet interactions will reveal individualized nutritional approaches to modulate the risk for AMD.

The objective of this study was to explore, in the Cfh null mouse, whether modulation of the dietary GI would alter the incidence of AMD. Wildtype or Cfh null 12‐week mice were fed high or low GI diets for 9‐months and were then evaluated for abnormalities in the retina or retinal‐pigmented epithelium (RPE). Wildtype mice showed no abnormalities on either diet. Surprisingly, Cfh null mice showed distinct abnormalities in the RPE when fed a low GI diet. Electron microscopy revealed the presence of vacuoles, disruption of the basal infoldings, and increased numbers of basal laminar deposits – all hallmarks of early AMD.

Conclusion

a Cfh/GI interaction led to development of AMD‐like features, paradoxically involving a low GI diet. This work was supported by intramural funds from the HNRCA and NEI RO1 21212.
Microbial metabolites produced by the colonic microbiota as drivers for immunomodulation in the host
Authors: Venema Koen
Published: June 2021

The colonic microbiota ferments dietary substrates that are not digested in the upper GI tract, such as complex carbohydrates. Fermentation of these leads to the production of short chain fatty acids (SCFA), which have been shown to have several beneficial effects on the host, including functioning as immune signaling molecules. For instance, butyrate has been shown to reduce inflammation in inflammatory bowel disease, possibly through its histone deacetylase inhibitory activity. Propionate has effects on cells in the adipose tissue. The SCFA are hypothesized to signal through G‐protein coupled receptors. However, we have shown that additional pathways must be active. Several tools were combined to study SCFA production by the colonic microbiota, including a sophisticated, in vitro model of the colon including a complex microbiota, 13C stable isotope technology, and reporter cell assays. The microbiota used originated from several different volunteers, e.g., different ages, healthy or diseased. We have shown that different microbiotas lead to production of different ratios of SCFA, and thus may be portrayed to have different health effects in these individuals. Using specific prebiotics, the activity of the microbiota can be directed towards production of certain SCFA, and through this the immunomodulation in the host can be influenced.
Consumer knowledge, attitudes and behaviors about foods that have immune benefits: focus on mushrooms
Authors: Tejera Christian , House Lisa A , Percival Susan S
Published: June 2021

Knowledge about what foods consumers believe are linked to immune benefits is scarce. To identify what foods individuals believe support immunity, and specifically whether mushrooms were among those foods, an online survey was developed and administered to 500 random consumers in the United States. In addition to gathering information on which foods participants identified as supporting immunity, information was collected on purchase decisions and reasons for, or against, purchase of mushrooms. Finally, demographics were collected to investigate if results varied by groups such as age, race, or gender. Participants were first asked to identify any foods that supported immunity from a list of 16 foods, including mushrooms. The two top choices were blueberries and oranges; selected 74% and 73% of the time, respectively. Mushrooms were selected 24% of the time. Additionally, when participants were asked if they were aware of any specific health benefits related to mushrooms, over half (60%) were unaware of benefits, 22% were unsure, and only 18% felt there were health benefits from consuming mushrooms. We know from studies performed in this lab that mushrooms have a beneficial effect on immune function of healthy people. With less than 20% believing that mushrooms have any health benefit at all, there is much that can be done to educate the public on the benefits of consuming mushrooms. Funding: SRIP, FL Ag Ex Sta
Natural killer cell cytotoxicity is reduced in aged female mice fed a high folic acid diet
Authors: Sawaengsri Hathairat , Wang Junpeng , Desautels Nellie , Steluti Josiane , Histed Alex , Smith Donald , Wu Dayong , Meydani Simin N , Selhub Jacob , Paul Ligi
Published: June 2021

Unmetabolized folic acid in plasma, which is indicative of high folic acid intake, is associated with reduced natural killer (NK) cell‐mediated cytotoxicity in elderly (>;60y) women. In this study, we determined if high folic acid intake affects NK cytotoxicity in aged female mice. Aged (16 months) female C57BL/6 mice were fed a control diet (1× RDA folic acid; 2mg/kg) or a high folic acid (HF) diet (20× RDA folic acid;HF 40mg/kg) for 3 months. Folate concentration and splenic NK activity were evaluated. Mice on HF diet had significantly higher unmetabolized folic acid (29.0 pmol/mL versus 0.6 pmol/mL) and formylated folate (19.0 pmol/mL versus 3.1 pmol/mL) (P<0.01). Spleen NK activity was determined as percent dead target cells at multiple effector: target (E:T) cell ratios, where the dead (not lysed) target cells were identified by flow cytometry. In HF mice, percent dead target cells were lower by 14% and 23% for (E:T) cell ratio 25:1 (P=0.03) and 12.5:1 respectively (P=0.036) than the control mice. Percent NK cell was lower by 21% in the spleen of the HF mice when compared to the control mice (P<0.01), but not percent of NKT cells, suggesting that the decrease in cytotoxicity of HF mice might be due to the reduction in the number of spleen NK cells. Further studies are required to understand the mechanism that underlies the HF‐induced reduction in NK cell number and cytotoxicity.

Grant Funding Source: HNRCA Pilot grant
Nutritional and Immunological Lessons Learned from the Porcine Genome
Authors: Dawson Harry D
Published: June 2021

Emerging evidence suggests that swine are a scientifically acceptable intermediate species (rodent‐human) for conducting scientific research relevant to humans. The swine genome has recently been sequenced; but, a large‐scale analysis of its content with respect to genes involved in nutrition or immunity has not been reported. To this end, a genome‐based, analysis of model suitability of pig was conducted. This analysis revealed that familial gene expansion, driven mostly by immune‐related genes, has occurred in pigs relative to humans at half the rate of mice to humans. Furthermore, pigs and humans have 3 fold less unique genes compared to mice. The number of pig‐human restricted orthologs is 5 and 8 fold greater than the number of mouse‐human or mouse‐pig restricted orthologs respectively. Among shared orthologs, protein sequence homology is higher between human and pig compared to human and mice. Furthermore, protein functional domain preservation between human and pig is nearly twice that of mouse to human and pig to mouse. This large‐scale comparative assessment of the pig, mouse and human genomes revealed an overwhelming similarity of swine to humans for most parameters. This work will contribute significantly toward using swine as models to promote both human and animal health particularly those with a nutritional and immunological component.
Human Airway Epithelial Cells and Alveolar Macrophages Coordinate the Innate Immune Response to Acute Ozone Exposure
Authors: Bauer Rebecca N , Duncan Kelly , Diaz‐Sanchez David , Hernandez Michelle , Jaspers Ilona
Published: June 2021

Inhaled oxidant pollutants damage airway epithelial cells (EC) and induce cytokines and damage associated molecular patterns (DAMPs) to alert local immune cells and initiate an innate immune response. We examined how the interaction between EC and alveolar macrophages (AM) modifies innate immune responses to acute O3 exposure. Healthy volunteers were exposed to 0.3 ppm O3 or air for 2 hours. Bronchoalveolar lavage (BAL) was collected 1 hour post‐exposure and assessed for cytokines and DAMPs (ATP and uric acid). AM from the BAL were stimulated ex vivo with ATP and assessed for cytokine production. To model the EC‐AM interaction in vitro, AM from healthy volunteers were co‐cultured with ECs (16HBEo‐cell line). Co‐cultures, AM alone, and EC alone were exposed to 0.4 ppm O3 or air for 4 hours and assessed for cytokine production 1 hour post‐exposure. We found acute O3 exposure in vivo induced the release of interleukin (IL)‐8 and DAMPs, such as uric acid. At basal levels ex vivo, O3 and air exposed AM produced similar amounts of IL‐8. With ex vivo ATP stimulation the O3, but not air, exposed AM had enhanced IL‐8 production, suggesting additional signals from the airway were necessary. Likewise, in vitro O3 exposure of AM alone did not induce IL‐8 production, whereas AM‐EC co‐cultures had robust O3 induced IL‐8 production. These results suggest the interaction between EC and AM is a key determinant of the innate immune response to O3.
Modeling Long‐term Host Cell‐Giardia lamblia Interactions in an in vitro 3D Cell Culture System
Authors: Fisher Bridget Sutton , Estrano Carlos , Cole Judith
Published: June 2021

The flagellated, unicellular parasitic protozoan, Giardia lamblia, induces diarrheal disease worldwide in humans and other mammals. When attached to the gastrointestinal tract, Giardia induces apoptosis of the epithelial cells, disrupts tight junctions between adjacent cells, and increases permeability of the intestinal barrier; however, the underlying cellular and molecular mechanisms of giardiasis are poorly understood. One of the major obstacles in ascertaining Giardia pathology is the lack of a functionally relevant, host‐specific model for studying the long‐term interaction of the parasite with the intestinal epithelium and lamina propria immune cells. Therefore, we have built an in vitro 3‐D model using the macrophage‐like cell line, IC‐21, and the human colorectal adenocarcinoma cell line, CaCo‐2 clone C2BBe1. In comparison to monoculture experiments, the 3‐D model better represents the cellular complexity and spatial architecture of the small intestine. Using Giardia strain WBC6, we have demonstrated the long‐term survival of trophozoites in the model over 21 days. Additionally, we were able to mimic some aspects of parasite pathology associated with infection, including increased apoptosis of epithelial cells through caspase‐3 activation, altered mitogen‐activated protein kinase (MAPK) signaling assessed by phosphoantibody cell‐based ELISA, and changes in epithelial cellular permeability. Supported by: University of Memphis Intramural Research Funding
Decreased SLPI levels in smokers is associated with increased inflammation and IAV infection
Authors: Meyer Megan , Bauer Rebecca , Brighton Luisa , Jaspers Ilona
Published: June 2021

We have previously shown that smokers have deficient innate immune response to influenza A virus (IAV) resulting in enhanced viral replication. Viral entry of IAV depends on proteolytic cleavage, and we have shown that the balance between proteases and antiproteases, such as secretory leukocyte protease inhibitor (SLPI) significantly affects viral replication. SLPI levels can be controlled transcriptionally and we have previously shown that the transcription factor Nrf2 positively regulates SLPI expression. In addition, the SLPI promoter contains ISRE regions, which may suppress transcription. We hypothesized that dysregulation of SLPI levels in epithelial cells from smokers enhances susceptibility to IAV. To test this hypothesis, apical washes and whole cell lysates from nasal epithelial cells from smokers and non‐smokers were analyzed for SLPI and STAT1 protein levels. At baseline, NEC from smokers had decreased SLPI and increased STAT1 levels. Moreover, apical washes from NEC of smokers resulted in enhanced cleavage and proteolytic activation of IAV, suggesting decreased SLPI activity. In addition, NEC treated with rhSLPI for 4h and infected with IAV for 24h, resulted in decreased HA gene expression and viral titers only in NEC from smokers. As such, dysregulation of SLPI at the levels of the epithelium may be key factor during IAV infection.
Control of VEGF‐Endothelial Response by ECM Stiffness
Authors: Derricks Kelsey E , Trinkaus‐Randall Vickery , Nugent Matthew A
Published: June 2021

Vascular endothelial growth factor (VEGF) drives both endothelial cell maintenance and normal/pathological angiogenesis. Recent research has suggested that mechanical signaling due to the stiffness of the cell contact surface may also have profound effects on endothelial cell behavior. To study VEGF signaling as a function of substrate stiffness, endothelial cells were cultured on fibronectin linked polyacrylamide gels and treated with VEGF. To analyze the response to VEGF we developed a custom MATLAB software script to analyze calcium flux movies. The script is able to individually identify cells, normalize each calcium signal value to the cell's individual background, and analyze the data. A variety of outputs are produced such as the magnitude of response, number of local maximums each cell experiences and clusters of coordinated cells. Utilizing this tool, we observed that VEGF‐stimulated intracellular calcium signaling varies in both magnitude and signaling kinetics depending on the stiffness of the substrate. At low VEGF concentrations we found maximal response (2–3.5 fold increases) at moderate stiffness whereas at high concentrations of VEGF the softest substrates showed the most intense response, but the response dissipated the fastest. We show that extracellular matrix stiffness can differentially drive endothelial response to VEGF. Supported by HL088572 and AHAF M2012014.
Modulating Myeloperoxidase‐Induced Endothelial Permeability by a Carbon Monoxide‐Releasing Molecule, CORM‐3
Authors: Patterson Eric K , Serizawa Fukashi , Capretta Alfredo , Fraser Douglas D , Potter Richard F , Cepinskas Gediminas
Published: June 2021

Previous work has demonstrated that CORM‐derived carbon monoxide (CO) supresses inflammation. However, the mechanisms are not well understood. Neutrophil (PMN)‐derived myeloperoxidase (MPO) is known to play a significant role in the pathophysiology of numerous inflammatory disorders through: 1) direct peroxidation and 2) producing hypohalous acids, particularly hypochlorous acid. Thus, an overwhelming MPO‐dependent production of oxidants in the sub‐endothelial space after PMN degranulation, contributes to tissue damage and propagating inflammation.

We hypothesized that CORM‐3‐derived CO interferes with MPO's catalytic activity, and thereby mitigates endothelial inflammatory damage.

In vitro assays were used to study the inhibition of MPO's catalytic activity. Further, live cell in vitro assays were used to investigate endothelial damage through measuring reactive oxygen species (ROS) and cellular permeability.

Our results indicate that CORM‐3 inhibits total MPO activity as well as MPO's peroxidation and halogenation cycles. Cellular assays determined that ROS production and cellular permeability are decreased when extracellular MPO is treated with CORM‐3. In all assays inactivated CORM‐3 (iCORM‐3) was significantly less effective than CORM‐3.

We conclude that CO may mitigate inflammatory damage partly through inhibition of MPO activity. Support: HSFO‐393 and LHRI IRF‐025–09
Post‐translational protein modification in autoimmune epithelitis
Authors: Rodriguez‐Rodriguez Mayra , Pérez‐Pérez Maria Elena , Badillo‐Soto Adriana , Bollain y Goytia Juan José , Pacheco‐Tovar Deyanira , Avalos‐Díaz Esperanza , Herrera‐Esparza Rafael
Published: June 2021

Sjögren's syndrome is an autoimmune epithelitis of salivary and lachrymal glands that results in Sicca syndrome. Citrulline is synthesised by the conversion of ornithine to arginine during urea formation, exists as free citrulline produced by NOS enzymes, and another form produced by post‐translational modification of proteins at arginine residues by peptidylarginine deiminases (PADs), which is important in autoimmunity. We investigate post‐translational reactions in salivary gland biopsies from 24 patients with primary Sjögren's syndrome and 24 controls using immunohistochemistry and a tag‐purified anti‐cyclic citrullinated protein CCP antibody to detect citrullinated peptides, PAD2 also was assessed. The present work demonstrated the weak presence of the PAD2 in some normal salivary ducts (10%), although PAD2 expression was increased considerably in Sjögren's patients. The presence of citrullinated proteins in the salivary tissues of Sjögren's patients strongly supports the in situ post‐translational modification of proteins in this setting. The mutual expression of CCP and PAD2 (70%) suggests that this post‐translational modification is enzyme‐dependent. In conclusion, patients with Sjögren's syndrome expressed the catalytic machinery to produce post‐translational reactions that may result in auto‐antigen triggering.

Grant: P/PIFI 2011–32MSU0017H‐06–01