bluuebunny/crossref_metadata_2025 huggingface dataset:
Estrogen increases the conversion of α‐linolenic acid to docosahexaenoic acid in ovariectomized mice
Authors: Mason Julie K , Kharotia Shikhil , Wiggins Ashleigh K.A. , Chen Jianmin , Thompson Lilian U.
Published: June 2021

n‐3 polyunsaturated fatty acids (PUFA), namely eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have beneficial health effects. The conversion of α‐linolenic acid (ALA) to EPA and DHA is low, especially in males compared to females and varies in the literature. Estrogen (E2) is suggested to affect ALA conversion but few studies have confirmed the E2 effect. Our objective was to determine the independent effect of E2 on n‐3 PUFA levels in serum and various tissues in female ovariectomized mice fed diets with low and high ALA. After 7 weeks exposure to an E2 pellet, mice were randomized to a corn oil‐based basal diet or an ALA‐rich flaxseed diet (FS) with or without removal of the E2 pellet for 4 weeks. E2 responsiveness was indicated by the 269% higher (P<0.001) uterus weight in E2‐ treated mice compared to those with E2 pellet removed. FS‐fed mice had higher proportions of ALA, EPA and DHA in total lipids extracted from serum, liver and brain. E2‐treated mice had higher serum (15.1%; P<0.001) and liver (18.2%; P<0.001) DHA but not brain DHA. EPA levels were not affected by E2. There was a diet × E2 interaction for ALA in the liver where the FS+E2 group had lower ALA compared to FS‐E2. In conclusion, E2 results in higher serum and liver DHA, does not affect brain DHA and does not affect EPA in any of the tissues measured. This finding may help explain the variability in ALA effect in studies involving males and females.

Grant Funding Source: NSERC
Expressional Regulation of the Leptin by n‐3 FA is Related to The Epigenetic Modification of its Promoter in Diet Induced Obese Mice
Authors: Fan Chaonan , Shen Wenwen , Wang Cui , Deckelbaum Richard , Qi Kemin
Published: June 2021

In this study, we investigated epigenetic modifications in the promoter region of the leptin gene and related effects of dietary n‐3 polyunsaturated fatty acids (n‐3 FA) in diet induced obese (DIO) mice. Male C57 BL/6J mice were fed with two high‐fat diets with different fat types – sunflower oil (n‐3 FA deficient) and fish oil, with a low‐fat diet ‐‐ sunflower oil as control. Compared to the mice fed with the control diet, expressions of leptin in plasma and epididymal fat were increased 8 fold in the n‐3 PUFA deficient DIO mice (P<0.01), and fish oil n‐3 FA in the DIO diet reversed the increased leptin expressions. The mean methylation fractions in most CpG sites of the leptin promoter were higher in the DIO mice (77% and 75%) than those in control (70%) with increasing binding of methylated CpG binding protein 2 by 40%. Also, increased H3 acetylation and decreased H4 acetylation at the leptin promoter were found in n‐3 deficient DIO mice, and the increased H3 acetylation was inhibited by dietary n‐3 FA. Methylation of Lys4 on H3 (H3K4) was reduced and that of Lys9 on H3 (H3K9) was increased in n‐3 deficient DIO mice as compared with control mice, and n‐3 FA in the DIO diet reversed the reduction of H3K4 methylation with no effects on H3K9 methylation. Therefore, epigenetic modification of the leptin promoter region may relate to expressions of leptin and this process may be regulated in part by n‐3 FA.

Supported by the Nutricia Research Foundation (2009–15 and 2010‐E4).

Grant Funding Source: Nutrition (ASN)
Investigation of metabolic adaptation in developing chicken embryo and post‐hatch chicks by blood metabolomics
Authors: Hu Qiong , Agarwal Umang , Bequette Brian J
Published: June 2021

This study investigated metabolic differences in chicken embryos and post‐hatch chicks from two egg sizes and broiler breeders of different maternal ages using a metabolomic profiling approach. Eggs from breeders of different ages (32 wk vs 51 wk, 63.2 ± 1.2 g) and eggs of two sizes laid by 45 wk old breeders (55.8 ± 1.2 g vs 67.7 ± 1.1 g) were incubated (n = 9 – 10) under standard conditions. Blood were collected on embryonic (e) days 14, 17, and 20, and on post hatch d1. Blood metabolites were extracted with methanol and derivatizatized prior to analysis by gas chromatography‐mass spectrometry. Compound identification was determined using the Fiehn metabolomics RTL library, and concentrations were calculated using Agilent data analysis software. Pathway and Multivariate data analysis were performed using MetaboAnalyst. Principle component analysis indicated that embryos from different size eggs on both e14 and post hatch d1 clustered separately. By contrast, clustering was not observed in embryos from different age breeders. Pathway analysis revealed differences (P<0.05) in metabolism involving gluconeogenic and ketogenic amino acids (glutamine, glutamate, threonine, glycine, and serine), linoleic acid, and glycerolipids in embryos of different developmental stages. Conclusively, our results demonstrate that embryo metabolism in later developmental stages differs depending upon breeder age and egg size.

Grant Funding Source: Maize grant‐University of Maryland
Plasma Branch Chain and Aromatic Amino Acid Levels are Associated with Insulin Resistance in Nonalcoholic Fatty Liver Disease (NAFLD)
Authors: Sunny Nishanth E , Lomonaco Romina , Petrocelli Robert , Bashir Usman , Egan Austin , Cusi Kenneth
Published: June 2021

Elevated plasma branch chain (BCAA) and aromatic (AAA) amino acids have been implicated in the etiology of insulin resistance (IR) and also in future diabetes. We tested the relationship of BCAA/AAA with metabolic parameters of insulin sensitivity and liver fat (LFAT), in patients with NAFLD. Fifty patients, 7 without NAFLD and 43 with NAFLD as determined by MRS (LFAT ≥5.5%) were studied. We measured: 1) Total body fat by DXA; 2) Hepatic IR index [HIRi] and muscle (Rd) insulin sensitivity; 3) Plasma BCAA/AAA by mass spectrometry. Patients with NAFLD had severe hepatic and muscle IR (by the euglycemic insulin clamp). Plasma BCAA/AAA were already ~20% higher (p<0.05) with an increase in LFAT from 5.5 to 10%, compared to patients without NAFLD. A further increase in LFAT (from 11% to >;50%) only resulted in a modest additional elevation in plasma BCAA/AAA. Despite this, LFAT showed correlations with leucine (r=0.39), isoleucine (r=0.44), valine (r=0.42), and tyrosine (r=0.50, all p<0.01). Increased plasma BCAA/AAA correlated strongly with liver (HIRi) and muscle IR, especially under insulin‐stimulation: leucine (r=0.69/−0.58), isoleucine (r=0.65/−0.55), valine (r=0.57/−0.52) and tyrosine (r=0.68/−0.46), respectively (all p<0.001). In conclusion, plasma BCAA/AAA are elevated in patients with NAFLD and are closely associated with hepatic and muscle IR. Tissue specific alterations in BCAA/AAA metabolism may contribute to the severity of IR in NAFLD. Support: Burroughs Wellcome Fund and the American Diabetes Association (K.C.)
LACTATING MOUSE MAMMARY GLAND EXPRESSES SECRETED MGAM WHICH ENABLES UNWEANED SUCKLING PUP STARCH DIGESTION
Authors: Lee Nichols Buford , Diaz‐Sotomayor Maricela , Avery Stephen E , Hadsell Darrel L , Yan Like , Hamaker Bruce R , Quezada‐Calvillo Roberto
Published: June 2021

All starch digesting activities are reported absent in suckling mice by jejunal mucosal enzyme assay, imunohistochemistry or Western blots. We confirmed that there was no maltase activity or brush border staining with Mgam antibodies at 13d and that these were detected at 25d only in WT. Last year we reported that at 13 d both Mgam null and WT suckling mice can digest amylase treated 13C‐starch derived α‐limit dextrins (LDx) to increase blood 13C glucose paradoxically in 13d Mgam null suckling mice lumenal contents were stained by specific Mgam antibodies.

Objective

Test lactating mouse mammary glands for Mgam and Si gene expression.

Methods

RNA was extracted from the mammary gland of 10 different strains of WT 30d pp lactating mice. After RT, the cDNA was amplified for Mouse Si (amplicons for exons 1 to 6 or exons 16 to 18) and Mgam (amplicons for exons 1 to 7 or exons 3 to 7).

Results

Only the Mgam Exon 3 to 7 primers produced 516 bp bands on the Gel.

Conclusion

we suggest that maltase activity is secreted in mouse milk which enables unweaned pup starch digestion well before brush border bound enzyme development. Splicoforms of the Mgam message, lacking the membrane binding exon 2 coding region, in lactating mouse mammary tissue suggest synthesis of a soluble enzyme activity. This was confirmed by the expression of the exon 2 deleted Mgam message in lactating mammary glands of 10 different mouse strains.
Casein and gluten‐derived opiate peptides affect cysteine uptake and redox status
Authors: Shah Jayni Jatin , Trivedi Malav , Hodgson Nathaniel , Deth Richard
Published: June 2021

Symptomatic improvement upon dietary exclusion of casein and gluten (GF/CF diet) has been reported for autistic patients. β‐casomorphin‐7 (βCM7) and α‐gliadin‐7 (αG7) peptides are formed during digestion of milk and wheat/rye, respectively, which can activate opiate receptors. Blood and urine samples from autistic and schizophrenic patients contain higher amounts of these peptides, suggesting a possible contribution to these disorders. Systemic oxidative stress and brain neuro‐inflammation have been described in autistic subjects, in association with significantly lower levels of the antioxidant glutathione (GSH). We demonstrate that αG7 and human and bovine forms of βCM‐7 potently inhibit cysteine uptake (a rate‐limiting step for GSH synthesis) in cultured cells, affecting intracellular thiol metabolite levels and decreasing GSH. Bovine βCM‐7 was more potent in inducing these changes. Different signaling pathways were involved in mediating temporal effects of these peptides. Gene expression and site‐specific CpG methylation analyses were also performed. These findings provide a novel mechanistic explanation for the benefit of a GF/CF diet for the treatment of autism and other inflammatory disorders.

Funding agency: Autism Research Institute
Energy drink use in military populations: an exploratory analysis
Authors: Delahanty Liam Francis , Jones Eric , Attipoe Selasi , Deuster Patricia , Jones Donnamaria , Stephens Mark
Published: June 2021

Objective

To describe prevalence and patterns of energy drink/energy shot (ED/ES) use in a cohort of United States (US) military members.

Methods

A cross‐sectional web‐based survey of ED/ES use among US military members.

Results

827 of 23,275 responded (response rate of 3.6%). Of those who responded, 53% reported consuming ED and 23% reported consuming ES at least once a month. Reasons for use included needing an energy boost (44%) and increasing mental alertness (36%). Nearly one in five (17%) respondents reported increased mental alertness following ED use. Increased heart rate (10%) was the most common side effect. One in five (19%) respondents mixed ED with alcohol at least once in the past 30 days. Most users (71%) reported being at least somewhat familiar with ED/ES ingredients, although many believed that ED/ES were not safe (29%) or were unsure of their safety (21%).

Conclusions

ED/ES use is common among US military members. Most respondents were familiar with ED/ES ingredients but were either unsure about safety or believed use to be unsafe. Many respondents endorse mixing ED with alcohol. Data from this cohort will be compared with an ongoing survey of ED/ES use in military communities to better understand the prevalence of ED/ES use in these populations with the goal of increasing consumer education to promote safe use.

Funding by Center Alliance for Dietary Supplement Research (NA91FD).
Effects of a novel dietary supplement on physical and mental function in middle‐aged adults: a double‐blind, randomized, placebo‐controlled trial
Authors: Wood Steven M , Poole Steve , Bartlett Mark , Mastaloudis Angela , Burke Doug
Published: June 2021

Aging is characterized by a gradual loss in physical and mental function starting at 30–40 years of age. The purpose of this study was to compare a novel combination of cordyceps sinensis, pomegranate extract, and panax ginseng (TRT) to control (CNTL) formula regarding physical, mental, and sexual desire.

Healthy males and female subjects (n=116) were recruited to participate in a prospective, double‐blind, randomized, placebo‐controlled trial that lasted 60 days. Subjects were randomized into a control (n= 56; CNTL) or treatment (n= 60; TRT) groups and stratified by age and gender. Mental (MCS) and physical (PCS) wellbeing were measured by validated questionnaires. Other measurements included blood chemistry measurements, skin carotenoid concentrations, and adverse events.

There were n=48 in CNTL and n=47 in TRT groups that completed the study. No differences were detected in the mean scores for (PCS=CNTL;−0.47, TRT;−0.08) or (MCS=CNTL;0.32, TRT;−1.98) and sexual desire between groups nor between age. There was a strong trend towards higher skin carotenoid concentrations in the TRT group (P=0.052). There was no difference between groups for adverse events or blood chemistry. The TRT formulation appears to be safe. We were unable to detect differences in physical, mental, and sexual desire as seen in animal studies presumably because this population was so healthy and didn't show an age related decline. There was an interesting finding that subjects consuming the TRT formulation experienced a trend towards higher skin carotenoid concentrations and warrants further investigation in a population which has age‐related decline in physical, mental, sexual decline, or experiencing greater oxidative stress.

Research was supported by Nu Skin Enterprises, Provo, Utah
Uracil accumulation induced by folate depletion is dependent on uracil DNA glycosylase (UDG) activity
Authors: Ma Hongzhi , Wu Yizhen , Simon Kirk , Ahmed Aqila , Cabelof Diane C
Published: June 2021

We have previously reported that folate depletion inhibits DNA repair capacity. Here we investigate the impact of folate depletion on DNA damage and repair to determine whether the response to folate depletion is cell‐type specific. We find that DNA base excision repair (BER) capacity and uracil accumulation is a function of the ability of the cell to initiate BER when folate is limiting. Folate depletion induces Uracil DNA glycosylase (UDG) and BER in mouse liver, but does not induce uracil accumulation. Accordingly, mouse BNL‐C2 liver cells grown in folate‐free media exhibit significant induction in UDG (88% increase, p<0.01) and BER (12% increase, p<0.01), with no change in uracil accumulation. However, MEFs grown in folate‐free media exhibit significant downregulation in UDG (40% decrease, p<0.01) and BER capacity (21%, p<0.01), and significant increase in uracil accumulation (30% increase, p<0.01). Likewise, homozygous deletion of UDG significantly increases the impact of folate depletion on uracil accumulation, demonstrating a direct role for UDG and BER initiation on uracil accumulation when folate is limiting. We are presently measuring expression of folate‐metabolizing enzymes to determine whether the BER and uracil accumulation phenotypes are related to cell type specific changes in folate metabolism when folate is absent.

Research support: The Ellison Medical Foundation
A comprehensive list of sociodemographic, dietary and lifestyle factors has limited predictive power in determining RBC folate concentrations maximally protective against neural tube defects
Authors: Hartman Brenda , Hanley Anthony J , Aufreiter Susanne , Colapinto Cynthia K , Tremblay Mark S , O'Connor Deborah L
Published: June 2021

Optimal maternal folate status is associated with a reduction in the occurrence of neural tube defects (NTD). Socio‐demographic and lifestyle factors collected as part of the Canadian Health Measures and the National Health and Examination Surveys account for less than 30% of the variation in RBC folate concentration, a measure of tissue stores. Using a comprehensive literature‐derived list of potential predictors of folate status, our aim was to re‐evaluate maternal socio‐demographic, dietary and lifestyle factors associated with a RBC folate maximally protective against NTD (≥906 nmol/L) to determine the feasibility of developing a tool that could be used by health clinicians to predict RBC folate ≥906 nmol/L. A Block food frequency questionnaire, and detailed additional questions on: dietary sources of folate; folic acid and B12 supplement intake; education; ethnicity; income; birth status; age, smoking; and alcohol use were collected from 101 women of reproductive age. Mean RBC folate was 849.9±264.6 nmol/L as determined by microbial assay; 39% had RBC folate concentrations ≥906 nmol/L. Only higher folic acid supplement intake had a positive association (OR=2.6; 95% CI 1.38, 4.9) with RBC folate ≥906 nmol/L while low alcohol intake was negatively associated (OR=0.3; 95% CI 0.09, 1.0). We conclude that use of a comprehensive list of factors did not improve the prediction of RBC folate protective against NTD.

Grant Funding Source: CIHR (218776), NSERC (46513), and Hospital for Sick Children RTC Matching Funds Program (BH)
Dietary intake and major food sources of vitamin B6 in Korean adults
Authors: Kim Young‐Nam , Cho Youn‐Ok
Published: June 2021

To determine vitamin B6 intakes and major foods sources of Korean adults, three consecutive 24‐hour recalls were collected from 162 healthy adults (20–64 y) living in Seoul and metropolitan area, Gumi, and Kwangju, South Korea, during November, 2011 – March, 2012. The mean vitamin B6 intake and average ratio of vitamin B6 to daily protein intake were 1.80 mg/day and 0.025 mg/g protein, respectively. Vitamin B6 intake of males was higher than that of females (P<0.001), but there was no significant difference in the ratio of vitamin B6 to protein intake by gender. Foods from animal and plant sources provided 29% and 71%, respectively, of the total vitamin B6 intake. The top 10 major dietary sources of vitamin B6 in these Korean adults were Korean Chinese cabbage kimchi, rice, dumpling, pork, rice cake, beef, mandarin orange, banana, chicken, and sweet potato. Only rice, pork, and banana are found in the top 10 sources indicated a decade ago. As for major dietary sources of vitamin B6, the top 10 foods provided 50% of vitamin B6 intake in the current study, whereas it was 64% of total vitamin B6 in Korean adults reported in 2001. Therefore, current dietary contributors of vitamin B6 in Korean adults are more varied compared to those of a decade ago. Supported by 2012 research fund of National Research Foundation of Korea (2012R1A1A2A04669745).
Effect of a flavonoid‐rich cacao extract on vascular reactivity in a mouse model
Authors: Castillo‐Hernández Karen Geneve , del Carmen Castillo‐Hernández María , Guevara‐Balcázar Gustavo , Coral‐Vázquez Ramón Mauricio
Published: June 2021

Background

Cardiovascular disease (CVD) is the second leading cause of death in Mexico in both women and men. Evidence based on epidemiological studies suggests that consumption of flavanolrich foods is inversely related to the development of CVD. It has been suggested that cacao flavonoids has positive effects on vascular system, and more specifically at the endothelial level.

Objectives

To determine whether a treatment based on a flavonoid‐rich extract from Theobroma cacao beans could contribute to decrease the number of vasoconstrictions and improve vasodilator response in old mice.

Materials and Methods

We conducted an experimental, comparative, cross‐sectional study. Firstly ethanolic extraction was performed from Mexican Theobroma cacao beans, which was used for the experiments. We made two groups of mice (treated, untreated). Vascular reactivity was assessed with two experimental strategies: 1) Quantifying the presence of coronary artery vasoconstriction in both groups and 2) Evaluating the relaxant response to acetylcholine in an organ‐isolated system.

Results

There were more vasoconstriction in the placebo group (no extract), and although there was no statistically significant difference (p = 0.4596), there was almost 50% less vasoconstrictions in mice treated with the extract. In evaluating the relaxant response to acetylcholine, the mice receiving the extract showed a relaxant concentration‐response that was significant at higher concentrations (p<0.01), unlike control mice.

Conclusions

Through the ethanolic extraction from Theobroma cacao beans we could obtain an extract rich in flavonoids. The administration of this extract improves endothelium‐dependent relaxant response in this animal model.

Source of research support:CONACYT
Manganese supplementation reduces monocyte adhesion to endothelial cells by reducing ROS and downregulating ICAM‐1 independently of MnSOD
Authors: Burlet Elodie , Jain Sushil K
Published: June 2021

Manganese (Mn2+ ) levels have been found to be lower in the plasma and lymphocytes of diabetic patients and in the heart and aortic tissue of patients with atherosclerosis. Studies in our laboratory have shown that Zucker diabetic fatty rats (ZDF) supplemented with Mn2+ had lower cholesterol (25%, p<0.05), ICAM‐1 (17%, p<0.04), and higher adiponectin (38%, p<0.01) levels in blood. Cell culture studies with Human Umbilical Vein Endothelial Cells (HUVECs) showed that Mn2+ supplementation reduces adhesion of monocytes to endothelial cells (ECs). This study examines the mechanisms by which Mn2+ supplementation decreases monocyte adhesion to ECs, a key event in the development of atherosclerosis. HUVECs were treated with Mn2+ (0, 5, 10 μM) for 24 hours, then exposed to high glucose (HG, 25 mM) for another 24 hrs. Results demonstrate that Mn2+ supplementation decreases ICAM‐1 surface (20%, p<0.05) and total expression (65%, p<0.005), MCP‐1 secretion (16%, p<0.05), and ROS levels (21%, p<0.02) in HG‐treated HUVECs. Silencing studies using siRNA against MnSOD showed that similar effects of Mn2+ on ICAM‐1 expression and ROS were observed in MnSOD knockdown HUVECs. This study suggests that Mn2+ supplementation can decrease monocyte adhesion to ECs, and thereby delay progression of atherosclerosis in diabetes, by decreasing ROS, MCP‐1 secretion, and ICAM‐1 expression, independently of MnSOD.

Grant Funding Source: NIDDK and Office of Dietary Supplements
Physiological Responses to Caffeine are Moderated by Sex and Pubertal Phase
Authors: Ziegler Amanda M. , Graczyk Adam , Bendlin Ashley , Vattana Karina , Sion Teresa , O'Leary Shannon , Temple Jennifer L.
Published: June 2021

Objectives

The purpose of the study was to determine whether sex differences in response to caffeine are related to puberty and menstrual cycle phase.

Methods

Male and female participants (n=101), ages 8–9 and 15–17, had heart rate (HR) and blood pressure measured every 10min for 1hr after drinking a beverage containing placebo, or caffeine (1 mg/kg and 2 mg/kg). Participants received each treatment once during the first three visits and again two weeks later. Post‐pubertal girls began the first three visits 5–9 days after menstruation and the final three visits 5–9 days after ovulation.

Results

Caffeine treatment increased systolic blood pressure (SBP) and decreased heart rate based on dose. Pre pubertal children had greater decreases in HR than post pubertal children at the 2 mg/kg dose. In post pubertal participants only, sex differences in HR occurred while females were in the luteal phase, but not during the follicular phase. There was also an interaction of sex, pubertal phase, and caffeine treatment on SBP. Specifically, SBP increased for post pubertal males at 1mg/kg and 2mg/kg caffeine doses, but SBP increased for post‐pubertal females only after 2mg/kg caffeine treatment and only during the luteal phase.

Conclusions

Our findings confirm previous studies that show that caffeine increases blood pressure and decrease heart rate. These effects are moderated by pubertal phase, sex, and menstrual cycle phase.
Changes in RAPD molecular marker polymorphism during Cordyceps sinensis maturation
Authors: Gao Ling , Yao Yi‐Sang , Wu Zimei , Zhu Jia‐Shi
Published: June 2021

Cordyceps sinensis (Cs) is a precious traditional Chinese herb, and is widely used for a variety of therapeutic purposes. With use of mycology and molecular biology approaches, scientists have identified >;30 genera of fungi and at least 6 genotypes of Ophiocordyceps sinensis from wild Cs. We reported maturational changes in chemical components, differential expression and functionality of Hirsutella sinensis (Hs) in Cs. We used a fuzzy molecular technique, RAPD, to detect global alterations of fungus expression‐related molecular marker polymorphism. Fresh Cs was collected in May for premature (stroma 1.5 cm on average) and maturing (stroma 4 cm on average) Cs, and in June for mature Cs (stroma 7 cm on average) from Kangding, Sichuan, China. Genomic DNA was isolated individually from caterpillar body, stroma and ascocarp of Cs, serving as the PCR templates, and 20 random primers were used for PCR‐RAPD. RAPD polymorphism changed in caterpillar body and stroma during Cs maturation, when number and density of the DNA bands were considered for RAPD analysis. The polymorphism was highly similar for mature stroma and ascocarp, but very different when comparing Hs and Cs samples. In conclusion, Cs is undergone a maturational change in expression of Cs component fungi in its caterpillar body and stroma. The polymorphism difference between Hs and Cs samples supports the multi‐fungi, micro‐ecology hypothesis of Cs.
Identification of resveratrol‐3‐β‐glucoside in California almond varieties
Authors: Xie Liyang , Bolling Bradley
Published: June 2021

Stilbenes have been identified in grapes, blueberries, peanuts and pistachios, but little is known about its abundance in other polyphenol‐rich foods, such as almonds. Therefore, the objective of this study was to screen Nonpareil, Butte and Carmel almond varieties from California for the presence of stilbenes (n = 3 samples/variety). Stilbenes were determined in ethanolic almond extracts by UPLC‐MS. Instrument conditions were optimized for quantitation of polydatin, piceatannol, resveratrol, pterostilbene, and oxyresveratrol. Polydatin, resveratrol‐3‐β‐glucoside, was identified in almond extracts, with mean contents of 7.19, 8.52 and 7.44 μg/100 g almond in Nonpareil, Butte and Carmel, varieties respectively. Among all varieties, polydatin was concentrated in almond skins, which accounted for 95.6–97.5% of total almond polydatin content. Other stilbenes were not detected in almond extracts. Therefore, almonds contain stilbenes in addition to the previously identified polyphenol classes of proanthocyanidins, hydrolyzable tannins, flavonols, and phenolic acids. This work was supported by the Almond Board of California.

Grant Funding Source: Almond Board of California
The natural carotenoid astaxanthin, a PPAR modulator, ameliorates hepatic steatosis in C57BL/6 mice
Authors: Jia Yaoyao , Lee Sung‐Joon
Published: June 2021

Astaxanthin (AX), a natural carotenoid abundant in seafood, has hypolipidemic activity. Previously, we reported that AX is a PPARα agonist and PPARγ antagonist in reporter gene assay and time‐resolved fluorescence energy transfer analyses, via direct binding to PPAR subtypes measured by surface plasmon resonance experiments, and significantly reduced cellular lipid accumulation in lipid loaded hepatocytes. To confirm the effects of AX in vivo, we fed C57BL/6 mice by standard and high fat diet (HFD) for 8 weeks to induce the hepatic steatosis, and then orally administrated with olive oil with (6 mg/kg body weight and 30 mg/kg body weight) or without AX for 8 weeks. AX feeding significantly improved liver histology of HFD fed mice and reduced the level of triglyceride both in plasma and in liver. Although AX administration has not changed the body and liver weight of the mice, the abdominal fat weight was significantly lower in high dosage of AX fed mice compared with the HFD fed mice. We concluded that AX reduces hepatic lipid accumulation in lipid‐loaded hepatocytes and HFD fed mice, and the effects of AX may via regulate the lipid metabolism in the hepatocytes and liver by the net effects of activating PPARα and inhibiting PPARγ simultaneously.
NIH/ODS Analytical Methods and Reference Materials (AMRM) Program: Accomplishments and Future Directions
Authors: Betz Joseph M , Saldanha Leila G , Cragg Gordon L , Sorkin Barbara C , Coates Paul M
Published: June 2021

Assuring the quality of natural products is a challenge to manufacturers, researchers, regulators, and consumers. Because biological materials contain complex mixtures of metabolites, they pose unique analytical challenges. Evaluation of the accuracy, precision and reproducibility of chemical measurements made in these complex matrices is vital. The AMRM Program provides tools to help scientists in the dietary supplement and nutrition communities address these challenges. Program elements include: 1) publicly available methods that have undergone formal validation studies (useful for facilitating method selection and for dispute resolution); 2) suites of certified matrix reference materials for use as method development and method evaluation tools; 3) certified calibration standard solutions for use in analyte identification and instrument calibration; 4) fee‐free laboratory quality assurance programs to assist laboratories in evaluating and improving method, analyst, and laboratory performance. The poster will provide an overview of accomplishments and resources made available by the program, and include future program directions.
Evaluation of Pumpkins as a novel source for α‐carotene
Authors: Kemp Michael , Dever Joseph , Thompson Amber , Metzger Brandon , Barnes David
Published: June 2021

α‐carotene has been identified as having anti‐oxidant potential, anti‐carcinogenic properties, and associations with reduced allcause mortality. Pumpkins have been reported to have high concentrations of α‐carotene. To confirm this, we examined the α‐carotene concentration in five different colored varieties of pumpkins: Rouge Vif D'Etampes (red), Wolf (orange), Long Island Cheesewheel (Pink), Musque de Provence (Green), and Valenciano (white). The closer a pumpkins skin colors was to the red spectrum the greater their total carotenoid content. However, the further away pumpkins were from the red spectrum the higher their α‐carotene content. The green Musque de Provence and pink Cheesewheel pumpkins also appears to be a better sources for alpha‐carotene because of a higher ratio between α and β carotene, which means excessive amounts of β would not have to be consumed to achieve a good intake of α. Finally, the pumpkins highest in α‐carotenoids have greater potential for activation of ARE promoter elements which are known to control anti‐oxidant genes, is in‐line with other research reports of enhanced antioxidant potential for α‐carotene. These results suggest green Musque de Provence and pink Cheesewheel pumpkins are good sources for α‐carotene.
Measurement of the whole blood transcriptomic signatures in healthy elderly subjects fed the probiotic bacteria Lactobacillus rhamnosus GG ATCC 53103 (LGG)
Authors: Solano‐Aguilar Gloria I , Molokin Aleksey , Botelho Christine , Fiorino Anne‐Maria , Chen Celine , Urban Joseph , Dawson Harry , Haverkamp Miriam , Wang Linda , Hibberd Patricia
Published: June 2021

Infectious diseases remain a considerable cause of morbidity and mortality worldwide particularly in the elderly. Feeding specific probiotic bacterial strains has been purported to exert human health benefits, but there are few comprehensive molecular studies of the effect of probiotics in healthy human subjects. Therefore, we investigated the effect of feeding a widely used probiotic to ten clinically healthy elderly invited to an open label study where participants consumed 1 × 1010 cfu of LGG continuously for 28 days. Transcriptomic signatures were compared using deep RNA sequencing (RNA‐seq) of 500ng of globin‐reduced RNA isolated from whole blood of subjects prior to (baseline) during (day 28), and 28 days after feeding of LGG ceased (day 56). Differentially expressed genes (DEG) with an absolute 1.5 fold difference were analyzed using Ingenuity Pathway Analysis (IPA). A total of 97 DEG were found at day 28 after feeding LGG. DEG were significantly enriched in functions related to cell death and survival, cellular growth and proliferation and cellular movement. Overall, the data indicated that LGG consumption modified whole blood transcriptomic expression in healthy elderly subjects. Measuring gene expression by RNA‐seq can be used to evaluate whether LGG impacts immune responsiveness to vaccines.
Lower plasma DHA and EPA and their related oxylipins are potential contributors to vascular disease in hemodialysis patients
Authors: Watkins Bruce A , Kim Jeffrey , Kaiser Shaun , Friedman Allon N , Li Yong , Pedersen Theresa L , Newman John W
Published: June 2021

Hemodialysis patients (HDP) have high blood pressure compared to healthy controls and greater incidence of chronic inflammation, higher C‐reactive protein and risk for cardiovascular disease. Moreover, HDP patients typically demonstrate unusually low plasma n‐3 PUFA levels. The purpose of our investigation was to characterize the oxylipins and endocannabinoids in female HDP (55.5 ± 14.2 y, 8.1± 8.4 y on dialysis) and healthy matched controls (54.8 ± 5 y). Plasma was extracted and subsequently analyzed using ultra performance liquid chromatography followed by electrospray ionization and tandem mass spectrometry for oxylipins and endocannabinoids, while RBC and plasma lipid fatty acids were analyzed by GC. HDP showed lower EPA and family related oxylipids 5‐ and 12‐HEPE and 14–15‐ and 17,18‐DiHETE in plasma compared to controls. The potent n‐3 vasodilators 17(18)–EpETrE and 19(20)‐EpDPE were lower in the HDP compared to the controls raising the possibility that hypertension in HDP could in part be due to reduced peripheral vascular relaxation. Further, the levels of 2‐OG, 1‐LG and 2‐LG were higher in HDP compared to the healthy controls. Herein, we report novel findings on oxylipins and endocannabinoids in female HDP that demonstrate differences compared to controls. This research will provide new opportunities to evaluate the role of dietary PUFA in clinical disease in kidney dialysis patients.
Reactive Oxygen Species and Electron Flow Are Needed to Oxidize Cytochrome c at the Methionine Residues
Authors: Aluri Hema S. , Chen Qun , Hu Ying , Lesnefsky Edward J.
Published: June 2021

Objective

We hypothesize that during oxidative stress, an increase in intra‐mitochondrial reactive oxygen species (ROS) generation coupled with electron flow into the cytochrome c (cyt c) segment of the electron transport chain (ETC) is required to modify cyt c at key methionine residues, including methionine 81 (Met‐81) that forms a neo‐peroxidase activity catalyzing the oxidation of cardiolipin.

Methods

Mitochondria were isolated from C57BL6 mice (MITO). MITO were incubated with specific substrates and inhibitors to isolate segments of the ETC: (1) succinate + rotenone; (2) succinate + rotenone + Thenoyltrifluoroacetone (TTFA) (3) succinate + rotenone + antimycin A (4) succinate + rotenone + azide. Following incubations, cyt c was immunocaptured and methionine sulfoxide [Met (O)], a marker of methionine oxidation, including at Met‐81, was assessed using immunoblotting, normalized to cyt c content.

Results

ETC Blockers incubated with MITO, succinate and rotenone
Conditions cyt c exposed to
fold increase compared to succinate + rotenone

Control: No blocker
none
1.0±0.0

Complex II: TTFA
ROS
1.03±0.38

Complex III: A.A
ROS
0.87±0.16

Complex IV: Azide
ROS, electron flow
3.5±1.1*

N=5–7; Mean ± SEM; one‐way ANOVA;

p<0.05 vs. no blocker

Conclusions

From the MITO incubations, it is clear that both ROS and electron flow into the cyt c segment are required for methionine oxidation and the likely formation of cyt c peroxidase. Supported by Research Service, Department of Veterans Affairs and the Pauley Heart Center of Virginia Commonwealth University.
Interaction of MCM7 and RACK1 for activation of MCM7 and cell growth
Authors: Zhang Xiyue , Yu Yan P , Luo Jian‐Hua
Published: June 2021

MCM7 is one of the pivotal DNA replication licensing factors in controlling DNA synthesis and cell entry into S phase. Its expression and DNA copy number are some of the most predictive factors for the growth and behavior of human malignancies. In this study, we identified that MCM7 interacts with receptor for activated protein kinase C 1 (RACK1), a protein kinase C (PKC) adaptor, in vivo and in vitro. The RACK1 binding motif in MCM7 is located at the amino acid 221–248. Knocking down of RACK1 significantly reduced MCM7 chromatin association, DNA synthesis and cell cycle entry into S phase. Activation of PKC by 12‐O‐tetradecanoylphorbol‐13‐acetate dramatically decreased MCM7 DNA replication licensing and induced cell growth arrest. Activation of PKC induced redistribution of RACK1 from nucleus to cytoplasm, and decreased RACK1‐chromatin association. MCM7 mutant that does not bind RACK1 has no DNA replication licensing nor oncogenic transformation activity. As a result, this study demonstrates a novel signaling mechanism that critically controls DNA synthesis and cell cycle progression.
Identification of PKM2 Interacting Proteins by Tandem Mass Spectrometry
Authors: Hwang Steven Ray , McDonnell Scott RP , Basrur Venkatesh , Conlon Kevin P , Fermin Damian , Murga‐Zamalloa Carlos , Elenitoba‐Johnson Kojo SJ , Lim Megan S
Published: June 2021

Pyruvate Kinase M2 (PKM2) plays a key role in aerobic glycolysis and the metabolic phenotype that is critical for cancer cell growth and proliferation. Recent studies have demonstrated that PKM2 may have nonmetabolic functions like regulation of gene transcription and protein phosphorylation. We hypothesized that liquid chromatography tandem mass spectrometry (LC‐MS/MS) “interactome” analysis of PKM2 will identify additional functional roles of PKM2. We carried out immunoprecipitation (IP) of DEL cells stably expressing FLAG‐tagged wild type (WT) PKM2 and the Y105F mutated PKM2 (glycolytically active) and subjected the IP to LC‐MS/MS. A total of 274 proteins were detected in the WT PKM2 pull down. There were 57 proteins unique to WT PKM2 relative to the Y105F PKM2 mutant. We carried out validation via IP and western blot for three proteins involved in RNA metabolism and gene regulation (HMGA1, RNF20, and PTBP1). Interaction of these proteins was predominantly detected with the WT isoform and not the Y105F isoform. DAVID analysis showed that WT PKM2 interacted more with proteins involved in nuclear functions such as RNA metabolism and gene expression than its mutated counterpart. Our results indicate that PKM2 may have a role in RNA metabolism and gene regulation through its interaction with HMGA1, RNF20, and PTBP1.

Funding support comes from the University of Michigan, Department of Pathology development funds.
mTOR inhibition leads to decrease of breast cancer progression in vitro via cytoskeleton remodeling
Authors: Cherednyk Olga , Khoruzhenko Antonina , Kosach Viktoria , Filonenko Valeriy
Published: June 2021

Kinase mTOR is one of the main links in signal transduction from variety of growth factors/hormones into the cell. Earlier it was shown its overactivation in numerous of cancers. mTOR inhibitors are regarded as antitumor drugs.

Immuunofluorescent analysis was applied to detect subcellular localization of mTOR in MCF‐7 cells and human breast tumor. The effect of rapamycin on cultured cells was tested by MTT‐test, migration, adhesion/spreading assay, zymography, actin detection with falloidin, confocal microscopy.

Cell behavior under the condition of inhibited mTOR activity was analyzed in vitro. It was revealed decrease of MMP‐9 activity, cell adhesion (up to 40%), cell spreading, migration in “wound healing” model (up to 80%). It was shown the apparent change in actin cytoskeleton organization in paranuclear space.

Detection of mTOR localization with anti N‐terminal antibodies revealed a stained network in cells. Confocal microscopy analysis shown a strong colocalization of mTOR and intermediated filaments in MCF‐7 and Hela as well at histological sections of normal and malignant breast tissue.

The effect of mTOR activity inhibition on cell behavior can be explained by its relation with cytoskeleton.
Bioenergetic characterization of adipogenesis impaired by inflammation
Authors: Esposito Debora , Wilson Mickey , Holway Haley , Komarnytsky Slavko
Published: June 2021

Adipocytes have been fundamental in identifying basic cellular mechanisms associated with diabetes and obesity, but their bioenergetics has not been fully understood. We measured changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) during differentiation of naïve and TNF‐α exposed 3T3‐L1 preadipocytes using Seahorse XF24 Extracellular Flux Analyzer, and correlated them to mRNA and protein markers of adipogenesis. Basal OCR and ECAR increased as the 3T3‐L1 cells underwent adipogenic differentiation from 110.7±26.5 to 901.1±86.9 pmol/min, and from 7.7±1.4 to 21.6±1.8 mpH/min, respectively. The complex V inhibitor oligomycin reduced OCR of the mature adipocytes to 63% of baseline rates, suggesting that 37% of cellular oxygen consumption was coupled to ATP synthesis. The combination of complex I and II inhibitors rotenone and antimycin reduced OCR to 16% of baseline, indicating that mitochondrial fraction accounted for 84% of total cellular respiration. Bioenergetic parameters of naïve and TNF‐α exposed preadipocytes and adipocytes responded differently to certain substrates (glucose, pyruvate, lactate, and palmitate), PPARα/γ activators, and β‐adrenergic agonists, highlighting intrinsic differences in maintaining energy homeostasis during adipocyte differentiation and possible molecular targets for nutritional preemption of metabolic disorders.
Alteration of endothelial CaMKII in AngII‐induced hypertensive mice
Authors: Charbel Chimene , Toussaint Fanny , Blanchette Alexandre , Ledoux Jonathan
Published: June 2021

Endothelial dysfunction found in AngII‐induced hypertension is characterized by an alteration of the delicate balance between reactive oxygen species (ROS) and nitric oxide (NO). Loss of endothelial Ca2+ homeostasis has also been shown in hypertension. Recent evidences suggest that CaMKII is activated by Ca2+ pulsars, a localized spontaneous Ca2+ signal and is involved in endothelial function. Since CaMKII is also sensitive to intracellular ROS, endothelial CaMKII and Ca2+ pulsars in an AngII‐induced hypertension mouse model was studied. Real‐time confocal imaging showed that acute exposure to AngII stimulated both Ca2+ pulsars and production of ROS. As expected, an increase in endothelial ROS production was found with dihydroethidium (DHE) in the AngII hypertensive compared to normotensive mice. Moreover, AngII‐induced hypertension appears to recruit activated CaMKII (T286) at the myoendothelial projection (MEP), sites of Ca2+ pulsars. Consistently, clustering of all endothelial CaMKII isoforms (α, β and δ) within the MEP has showed a similar distribution. Our data suggest that CaMKII activity is modulated by an increased ROS production and Ca2+ pulsars stimulation in AngII‐induced hypertensive mice. Supported by CIHR, FRQS, CFI and HSFC.
Dynein‐mediated Lysosome Trafficking in Autophagic Flux of Mouse Coronary Arterial Myocytes
Authors: Xu Ming , Li Xiaoxue , Zhang Yang , Li Pin‐Lan
Published: June 2021

Dynein is a motor protein in a variety of mammalian cells that regulates lysosome trafficking. The present study hypothesized that dynein‐mediated lysosome trafficking contributes to autophagic flux, an important autophagy maturation process in coronary arterial myocytes (CAMs) and that nicotinic acid adenine dinucleotide phosphate (NAADP)‐induced lysosome Ca2+ release regulates dynein‐mediated lysosome trafficking and autophagic flux. Pretreatment of CAMs with EHNA (erythro‐9‐(2‐hydroxy‐3‐nonyl)adenine), an dynein inhibitor or overexpression of dynamitin that disrupts the dynein complex enhanced 7‐ketocholesterol (7‐Ket)‐induced expression of autophagic marker LC3B by 90.3 % and 82.5%, respectively. Similarly, EHNA and dynamitin cDNA increased the cellular level of p62, a selective substrate for autophagy, by 58.7 % and 52%, respectively. Confocal microscopy demonstrated that LC3B colocalization with Lamp‐1, a lysosome protein increased upon 7‐Ket and that their colocalization coefficient was reduced by 57.3 % and 54.1%, respectively with pretreatment of CAMs with EHNA or dynamitin cDNA. By flow cytometry, it was found that both EHNA and dynamitin cDNA increased 7‐Ket‐induced formation of autophagosomes (APs), but reduced the number of autophagolysosomes (lysosome‐autophagosome fused, APLs) in CAMs. In addition, 7‐Ket was found to increase lysosomal Ca2+ release as detected by colocalization of Ca2+(by Fluo4) and lysosome staining (by Rho) in living CAMs, which was blocked by 92.7 % when these cells were treated by NAADP inhibitors, NED‐19 or PPADS. These results suggest that atherogenic stimulus, 7‐Ket can enhance autophagy in CAMs and this 7‐Ket‐induced autophagy is associated with dynein‐mediated lysosome trafficking (supported by NIH grants HL057244, HL091464 and HL075316).
Testosterone induces activation of the extrinsic apoptotic pathway in VSMC by mechanisms involving ROS generation
Authors: Lopes Rheure , Neves Karla , Chignalia Andréia , Zanotto Camila , Pestana Cézar , Curti Carlos , Tostes Rita
Published: June 2021

The mechanisms by which testosterone (testo) affects the cardiovascular system may involve reactive oxygen species (ROS). ROS activate cell death signaling pathways. We hypothesized that testo induces ROS generation via mitochondria, culminating in caspase 3 activation and apoptosis. Cultured vascular smooth muscle cells (VSMCs‐mesentery of Wistar rats) were pre‐treated with flutamide(flu,10−5M), CCCP(10−6M), MnTmPyP(3×10−5M), ZIETD( 10−5M) or vehicle and then stimulated with testo(10−7M) (2–12h). ROS was measured by lucigenin and DCF, apoptosis by calcein and annexin V, procaspases/caspases 3/8 expression by immunoblotting. Cytochrome c, Bax and Bcl‐2 by immunoblotting of cytoplasmic and mitochondrial fractions. Testo (2h) induced ROS generation [162.6 ± 16 vs. 100], an effect abolished by CCCP (90.4 ± 15), MnTmPyP (26.2 ± 11.6) and flu (122.7 ± 10.6). Testo (6h) induced caspase 3 activation (166.2 ± 19 vs 100), which was abolished by flu (122.7 ± 10.6), MnTmPyP (61.8 ± 2,5), CCCP (71.6 ± 13,2) and Z‐IETD (125.1 ± 6). Testo (6h) increased annexin V fluorescence [197.6 ± 21,5 vs. 100] and decreased calcein fluorescence [34.4 ± 6.4 (2h) vs. 100]. Bax/Bcl‐2 ratio was reduced and there was no cytochrome c translocation in response to testo. Testo (6h) induced cleavage of procaspase 8 [161.1 ± 13.5 vs 100] and increased FasL [3.6 ± 1.2 vs 0.7 ± 0.5] and TNF [1.7 ± 0.4 vs 0.3 ± 0.1] gene expression, an effect abolished by CCCP, MnTmPyP and flu. In conclusion, testo induces apoptosis in VSMC by mechanisms involving ROS generation via mitochondria, androgen receptor and the extrinsic apoptotic pathway. Financial Support: CNPQ, FAPESP.