bluuebunny/crossref_metadata_2025 huggingface dataset:
Measurement of vitamin K homologues in biological fluids and tissues by APCI LC/MS
Authors: Leonard Scott William , Barker Tyler , Mustacich Debbie J , Traber Maret G
Published: June 2021

Vitamin K (K) is a family of molecules that contains a 2‐methyl‐1,4‐napthoquinone nucleus, including phylloquinone (K1) with a isoprenyl side chain, menaquinones with unsaturated isoprenyl side chains (K2; MK‐n) and menadione (K3; MK‐0). Here we describe a sensitive and specific liquid chromatography mass spectrometry (LC/MS) method to measure K1, K2 (MK‐4 & MK‐7), and K3 in biological fluids and tissues. For all assays a Micromass ZQ 2000 MS detector coupled to a Waters 2695 HPLC was used. For K1 & K2 analysis an isocratic method consisting of 100% methanol delivered at 1.0 mL/min for 30 min was used. For K3, a gradient of 50–90% methanol delivered over 30 min at 0.2 mL/min was used. Internal standards of ubiquinone‐4 and ‐2 were used for K1/K2 and K3, respectively. Calibration curves for the K forms generated a linear response from 1 to 50 nM, with a lower limit of detection of 0.2 nM (S/N of 3/1). K1 was detectable in human plasma, but MK‐4 was not. In livers from male Sprague‐Dawley rats consuming a K3‐containing diet, MK‐4 was detectable but K1 was not; large amounts of K3 were present in the urine. LC/MS with an APCI source is sensitive and specific enough to measure all of the expected vitamin K homologues in biological samples. This work was funded in part by the USDA, the NIH, and USANA.
Influence of dietary weight‐loss on vitamins D and K in obese post‐menopausal women
Authors: Shea M. Kyla , Nicklas Barbara J. , Houston Denise K. , Saltzman Edward , Kritchevsky Stephen B. , Booth Sarah L.
Published: June 2021

Obese persons are at risk for fat‐soluble vitamin (FSV) insufficiency. It is also plausible that weight loss is associated with change in FSV status. Hence, our aim was to examine associations between change in weight status and change in vitamin D and K status in 89 post‐menopausal obese women (mean±SD BMI=33.1±3.6kg/m2, age=58±6y), following a hypocaloric weight‐loss program. All women received 200IU/d vitamin D3 and 80 mcg/d vitamin K1. On average, baseline serum 25‐hydroxyvitamin‐D (25(OH)D) and vitamin K1 concentrations were within normal ranges (51±18nmol/L, 1.3±1.0nmol/L respectively). After 20‐weeks, women lost 11.1±5.3% of initial body weight and 8.0±3.2kg of fat mass. 25(OH)D increased by 10±14nmol/L (p<0.01, season‐adjusted), but serum vitamin K1 did not change (p=0.85, triglyceride‐adjusted). Using linear models, with change in FSV status as outcomes and change in weight‐status as primary exposures in separate analyses, there was no association between change in 25(OH)D and change in weight, BMI, or fat mass [unstd B(p‐value)= −0.51(0.15), −0.98(0.32), −0.60(0.19) respectively] or between change in serum K1 and change in weight, BMI, or fat mass [unstd B(p‐value)= −0.04(0.17), −0.10(0.16), −0.06(0.10) respectively]. Our data do not suggest moderate weight loss is associated with change in serum measures of vitamin D or K. Supported by NIA R01AGDK20583, P30AG21332 & USDA ARS #58‐1950‐7707.
Investigation of ω‐hydroxylation of tocopherols by CYP4F2 and by rat liver slices
Authors: Farley Sherry M. , Leonard Scott W. , Mustacich Debbie J. , Labut Edwin M. , Traber Maret G.
Published: June 2021

The ω‐hydroxylation of 2S‐α‐tocopherol (2S, 4′RS, 8′RS α‐tocopherol) and deuterated (d2)‐ γ‐tocopherol (d2‐γ‐T), relative to d6‐RRR‐α‐tocopherol (d6‐α‐T) was studied. Rat liver slices were incubated with d6‐α‐T, d2‐γ‐T, or 2S‐α‐T and their metabolism to 13′‐OH was measured by LC‐MS/MS. Although uptake of each form by liver slices was comparable, 2S‐α‐T and d2‐γ‐T were more readily metabolized to 13′‐OH (39 ± 15 pmol/g and 42 ± 5, respectively, p<0.01), than was d6‐α‐T (17 ±2). When these tocopherols were incubated with microsomes expressing human recombinant CYP4F2, there was greater production (p<0.001) of d2‐γ‐T‐13′‐OH (0.36 ± 0.05 pmol 13′‐OH/pmol CYP4F2) compared to d6‐α‐T‐13′‐OH (0.08 ± 0.02); however, there was no preference for 2S‐α‐T over d6‐α‐T. These data indicate that CYP4F2‐mediated ω‐hydroxylation does not differentiate between side chain stereoisomers and that structural differences in the chroman ring are more influential. This study also suggests that other factors present in liver slices, such as selectivity of α‐T transfer protein, are responsible in regulating metabolism of natural versus synthetic α‐T.

Grant Funding Source: NIH, ODS, Archer Daniels Midland, and USDA
Oxidative stress status of high prolific sows during pregnancy and lactation
Authors: Berchieri‐Ronchi Carolina Bragiola , Zhao Yan , Correa Camila Renata , Ferreira Ana Lucia dos Anjos , Yeum Kyung‐Jin , Kim Sung Woo
Published: June 2021

Sows are highly prolific with insufficient nutrient intake during late pregnancy and lactation periods causing a severe catabolic condition. Antioxidant status and oxidative DNA damage were determined in blood samples collected during the course of gestation and lactation in multiparous sows (parity 3 and 4). Blood samples were collected from sows (n=5) at d 30, 60, 90 and 110 of pregnancy, d 3, 10, and 18 of lactation, and d 5 postweaning. Lymphocytes were isolated from the fresh blood and cryopreserved in each time point. Lymphocyte DNA damage was analyzed by alkaline single cell gel electrophoresis (comet assay) to determine the single and double strand brakes, plasma total antioxidant performance (TAP) using fluorometry and endogenous antioxidant by a HPLC system with UV detection. The comet assay showed lower (p<0.01) endogenous DNA damage at d 30 of pregnancy (21%) as compared with those of the other time points (38 to 47%). On the other hand, TAP was lower (p<0.05) during d 30 to 90 of pregnancy as compared with that of d 10 of lactation. Plasma α‐tocopherol concentration was lower (p<0.05) at d 110 of pregnancy as compared with d 30 of pregnancy. The current study indicates that there is a systemic oxidative stress during the pregnancy and antioxidant nutrients can be reduced in the mother pig during the end of the pregnancy and that oxidative DNA damage can be elevated during the pregnancy, which can be sustained until the end of lactation period.

[Supported by CAPES 0635‐09, NCPC 09M69MF1, USDA 1950‐51000‐065‐08S]
Effect of long‐term Western style diet and folate supplementation on aortic response and hepatic C‐reactive protein level in C57BL/6 mice
Authors: Kong Eunhee , Hasan Syeda Tahira , Jang Hyeran , Zimmerly Ella May , Choi Sang Woon , Meydani Mohsen
Published: June 2021

Dietary folate and Western style diet (WD) are associated with the risk of CVD. After mandatory folate fortification was implemented, folate intake and blood folate concentrations in the US population have dramatically increased. We investigated vascular reactivity and systemic inflammation status from various long term (18 mo) folate intake levels (replete, 2mg/kg diet; deplete, 0.23mg/kg; and supplemented, 8 mg/kg) in C57BL/6 mice fed either control diet (AIN‐76A) or WD. Vascular reactivity of aortic rings of mice was measured by a multi‐chamber myograph. Following contraction of aortic rings with 0.3μmol/L phenylephrine, we measured the relaxation in response to acetylcholine (ACH; 0.01–10μmol/L). Hepatic C‐reactive protein (CRP) was measured by ELISA. Our results show that mice fed WD had less relaxation response to ACH, and this response was independent of folate intake. Folate supplementation reduced aortic ring relaxation by 21% compared to control group and by 30% in mice fed WD supplemented with folate compared to WD control. The level of CRP, regardless of folate levels in the diets, was higher in mice fed WD (100.77 ± 14.26 vs 75.38 ± 6.71, % ± S.D, p<0.05). Long‐term WD is associated with increased systemic inflammation as evident from increased liver CRP levels, and long‐term inclusion of folate in WD may have negative effect on vascular reactivity. Supported by USDA agreement #58‐1950‐7‐707
Tomato extract supplementation results in a preferential accumulation of hepatic phytoene and phytofluene and decreased plasma total cholesterol levels in high fat diet fed rats
Authors: Melendez‐Martinez Antonio J , Wang Yang , Mein Jonathan R , Wang Xiang‐Dong
Published: June 2021

We showed previously that dietary supplementation of tomato extract (TE) provides additional anti‐inflammatory potency as compared with lycopene (LY). In this study, we examined phytoene (PT), phytofluene (PTF) and lipid profiles in rats fed either control diet (35% fat, CD) or high fat diet (71% fat, HFD) with or without TE supplementation for 6‐wk. We found that TE supplementation results in a significantly higher accumulation of both PT and PTF than LY in livers of both groups. We also found that the hepatic levels of total cholesterol (TC), total triglycerides (TG) and free fatty acids (FFA) were significantly higher in HFD group as compared to the CD group. Interestingly, TE supplementation resulted in a further increase of hepatic TC, TG and FFA in the HFD group. However, TE supplementation in both CD and HFD groups significantly decreased the concentration of plasma TC. These data suggest that TE supplementation may decrease plasma TC associated with high dietary fat intake by regulating TC, TG and FFA in the liver. Moreover, TE supplementation results in a preferential accumulation of hepatic PT and PTF suggesting further investigation into their biological function(s). (Supported by NIH grant R01CA104932;USDA grant 1950‐51000‐064S;JCI‐2008‐01767 and JC‐2009‐00176).
Lifetime Dietary Exposure To Soy Isoflavones Is Beneficial To Prostate And Testicular Health In 100 Day‐Old Male Long‐Evans Rats
Authors: Blake Crystal , Hansen Tevor , Hogen Benjamin , Bourgeois Isaac , Lephart Edwin D
Published: June 2021

Consumption of soy isoflavones appears to be beneficial for several health parameters. We examined the effect of dietary exposure (from conception) of soy isoflavones on body weight (BW), white adipose tissue (WAT), prostate weight, and testicular weights (TW) and for several morphometric parameters. During pre‐ and postnatal development male rats were exposed to either a phytoestrogen‐rich (PR) or phytoestrogen‐low (PL) diet. At 100 days of age the male rats were sacrificed, tissues weighed, processed and analyzed by histology methods. BW and prostate/BW ratios were slightly decreased in the PR vs. PL groups (but were non‐significant). WAT/BW ratios were significantly less in the PR vs. the PL fed animals. Additionally, the percentage of invaginations of prostate glandular tissue was significantly greater in the PR vs. PL males. TW and TW/BW ratios were significantly greater in the PR vs. PL fed animals. Sertoli cell area was significantly greater in the PR vs. PL animals which accounted for greater seminiferous tubule lumens and seminiferous tubule areas in the PR vs. PL groups. These data indicate that consumption of a soy isoflavone rich diet appears to be beneficial in decreasing prostate glandular tissue and WAT deposition while increasing testicular weight, Sertoli cell area and seminiferous tubule volume which suggest a positive influence on reproductive capacity in males. (Grant Support: USDA 2004‐01811)
Curcumin modulates hemeoxygenase‐1, eNOS, and endothelial cell cycle progression
Authors: Jackson Steven J. T. , Venema Richard C. , Murphy Laura L. , Singletary Keith W. , Young Andrew J.
Published: June 2021

Curcumin (CMN), a component of turmeric spice that imparts flavor and color to curry, is a known anti‐inflammatory and angiogenesis‐suppressing bioactive agent. Here, we provide the first evidence that CMN inhibits proliferation of endothelial cells, while modulating hemeoxygenase‐1 (HO‐1) and eNOS protein expression. Exposure of non‐confluent bovine aortic endothelial (BAE) cells to CMN, at concentrations as low as 2 μM, resulted in suppressed proliferation, upregulated HO‐1, and downregulated eNOS within 24 h. In confluent cells, HO‐1 protein was similarly upregulated, while eNOS expression was unaffected by CMN. Concentrations of CMN in excess of 5 μM triggered G2/M cell cycle block in non‐confluent BAE cells, while 15 μM CMN clearly disrupted microtubule polymerization. Taken together, these findings indicate differential effects of CMN in cycling vs. noncycling endothelial cells, with potential dose‐dependent implications for wound healing related to endothelial tissue growth and repair. (Supported by USAMRMC)
Oxidation of energy substrates in rat brown adipose tissue
Authors: Xi Pengbin , Jiang Zongyong , Dai Zhaolai , Li Xilong , Yao Kang , Jobgen Wenjuan , Satterfield M. Carey , Wu Guoyao
Published: June 2021

This study determined the oxidation of glucose, fatty acids and amino acids in BAT of adult rats. BAT was isolated from 12 week‐old Sprague‐Dawley male rats and incubated at 37oC for 2 h in medium containing 1.25% bovine serum albumin, 0.1 nM insulin, plasma concentrations of amino acids, 5 mM glucose, and one of the following tracers: D‐[U‐14C]glucose, L‐[U‐14C]Arg (0.5 mM), L‐[U‐14C]Gln (0.5 mM), L‐[U‐14C]Leu (0.5 mM), L‐[1‐14C]Leu (0.5 mM), or [1‐14C]oleic acid (0.2 mM). Incubations were initiated by addition of BAT (~60 mg). 14C‐radioactivity was measured using a liquid scintillation counter. Values are means ±SEM, n = 8. There was no generation of CO2 from arginine in BAT. Rates of 14CO2 production from 5 mM D‐[U‐14C]glucose, 0.5 mM L‐[U‐14C]Gln, 0.5 mM L‐[U‐14C]Leu, and 0.2 mM [1‐14C]oleic acid in BAT were 2172±131, 915±84, 1018±96, and 46±3 nmol/2 h per g tissue, respectively. Rates of net transamination, net α‐ketoisocaproate release and oxidative decarboxylation of L‐[1‐14C]Leu in BAT were 614±51, 306±25 and 312±29 nmol product/2 h per g tissue, respectively. These results indicate that glucose, glutamine, leucine and fatty acids at physiological levels were oxidized in BAT as major energy substrates. Supported by grants from USDA, AHA and NSFC.
INGESTION OF DEOXYNIVALENOL REDUCES DIET‐INDUCED OBESITY IN THE MOUSE
Authors: Flannery Brenna , Amuzie Chidozie , Ulrich Allison , Romsos Dale , Pestka James
Published: June 2021

Deoxynivalenol (DON) is a Fusarium mycotoxin that has been long known to suppress weight gain of monogastric animals. We tested the hypothesis that DON consumption will reduce weight and adiposity in diet‐induced obese (DIO) mice. Female B6C3F1 mice were fed 60% kcal high fat diet (HFD) for 8 wk to induce obesity. They were then regrouped and fed HFD containing either 0, 2, 5,10 or ppm DON for an additional 8 wk. Animals fed HFD containing 5 ppm and 10 ppm DON exhibited reductions in body weights of 16% and 23%, respectively, while periuterine fat pad weights of the DON 10 ppm HFD group were reduced by 40%. Insulin like growth factor acid labile subunit (IGFALS) was reduced by 20 and 42% in the 5 and 10 ppm DON‐fed mice, respectively. In subsequent experiments, we determined that DIO mice fed DON exhibited reduced food intake and that these animals regained weight upon removal of DON from the diet. Taken together, our data indicate that DON attenuated weight and adiposity in DIO mice in a reversible manner and that this corresponded to both decreased food intake and deregulation of the growth hormone axis.

This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 59‐0790‐4‐119 and by PHS Grant ES03358
Greater fruit and vegetable intake is associated with increased bone mass in older Puerto Ricans
Authors: Bhupathiraju Shilpa N , Lichtenstein Alice H , Dawson‐Hughes Bess , Tucker Katherine L
Published: June 2021

Puerto Ricans, a group with documented health disparities, are the largest Hispanic sub‐group in Massachusetts. Yet, little is known about diet and bone health in this high risk population. We determined the association of fruit and vegetable (FV) intake (servings/day) with bone mineral density (BMD) among 613 Puerto Rican adults, aged 45–75 years. BMD at the lumbar spine (L2‐L4) and proximal femur were measured using dual energy X‐ray absorptiometry. FV intake was assessed using a food frequency questionnaire. Multiple linear regression showed significant positive associations of FV intake with BMD of the femoral neck (β=0.011, P=0.004), trochanter (β=0.010, P=0.009), and lumbar spine (β=0.015, P=0.01). Analyses were adjusted for age, sex, BMI, smoking, drinking status, season of BMD measurement, energy intake, calcium and vitamin D intake, servings of meat and fish, current estrogen use, physical activity, diabetes, blood pressure, and household income. The observed positive association between FV intake and BMD supports findings in other populations adding support to the notion that FV are important to bone health. Compounds and nutrients in FV can potentially neutralize acid production without depending on the buffering effects of bone minerals. Further, their antioxidant and anti‐inflammatory properties may also play an important role.

Grant Funding Source: NIH P01 AG023394 and USDA ARS contract 58‐1950‐7‐707
Fruit juice consumption decreases the proportion of children with inadequate intakes of key nutrients: NHANES 2003–2006
Authors: Zanovec Michael , Fulgoni Victor L , O'Neil Carol E , Nicklas Theresa A
Published: June 2021

Fruit juice (FJ) consumption has been under scrutiny despite its nutrient profile. NHANES (2003–2006) data were used to compare the proportion of children ages 2–18 years with intakes of selected vitamins/minerals below recommended levels among consumers (n = 3,976; 51% females) and non‐consumers (n = 3,274; 49% females) of FJ. Using the National Cancer Institute method for usual intake estimation, the percentage of the population with intakes less than the Estimated Average Requirement (EAR) was determined; for nutrients with an Adequate Intake (AI) we also compared the percentage of the population that consumed greater than the AI. A Z‐statistic for differences in population proportions was used to determine significance (p < 0.05). Compared to FJ consumers, a significantly higher percentage of non‐consumers had intakes below the EAR for vitamin A (24.4 ± 2.5 vs. 42.2 ± 2.5 %), vitamin C (0.1 ± 0.2 vs. 38.9 ± 4.1 %), folate (8.8 ± 1.5 vs. 22.1 ± 2.4 %), phosphorus (11.6 ± 2.1 vs. 21.3 ± 2.6 %), and magnesium (25.8 ± 1.7 vs. 46.1 ± 2.0 %). A greater percentage of FJ consumers exceeded the AI for calcium (51.3 ± 2.9 vs. 34.1 ± 2.3 %) and potassium (2.4 ± 0.5 vs. 0.5 ± 0.2 %) as compared to non‐consumers. Males and females benefited similarly from FJ consumption. FJ consumption improves nutrient adequacy and should be encouraged. (Supported by USDA and Juice Products Association)
Improved nutrient intake and diet quality with 100% fruit juice consumption in children: NHANES 2003–2006
Authors: O'Neil Carol E , Fulgoni Victor L , Zanovec Michael , Nicklas Theresa A
Published: June 2021

Fruit juice (FJ) consumption has recently been viewed as a sweetened beverage with little regard to its nutrient contribution to the diet. NHANES, 2003–2006, data were used to examine the association of 100% FJ consumption with nutrient intake and diet quality in children ages 2–5 y (n=1,665) and 6–12 y (n=2,446). Mean usual intakes, standard errors, and regression analyses (juice independent variable; vitamins/minerals and Healthy Eating Index‐2005 [HEI] were dependent variables) were determined using sample weights. Mean daily intake of 100% FJ was 5.76 ± 0.17oz (2–5 y) and 2.59 ± 0.37oz (6–12 y); 29% (2–5 y) and 43% (6–12 y) did not consume 100% FJ on the day of the recall. In children 2–5 y, magnesium (p=0.004), potassium (p<0.001), vitamin C (p<0.0001), and HEI (p<0.0001) (specifically whole fruit and whole grain) increased linearly and added sugars decreased linearly (p<0.0001) with higher levels of FJ consumption; in children 6–12y, magnesium (p<0.0001), calcium (p<0.0001), potassium (p<0.0001), dietary fiber (p<0.0001), vitamin C (p<0.0001) and HEI (p<0.0001) increased linearly and added sugars decreased linearly (p<0.0001) with higher intakes of FJ. On average, 100% FJ consumption did not exceed the American Academy of Pediatrics’ recommendation for children and was associated with increased consumption of key nutrients and better diet quality in children.

Funding: USDA and Juice Products Association.
Rab7 CMT2B Mutants Alter function in Endosomal Transport, Growth Factor Signaling and Regulatory Protein Interaction
Authors: Jim Patricia , Mukherjee Sanchita , Basuray Soumik , Romero Elsa , Contreras Chris , Wandinger‐ness Angela
Published: June 2021

Rab7 regulates transport and degradation on the late endocytic pathway by orchestrating interactions with motor proteins, proteasome, lipid kinases and phosphatases. Four different point mutants of Rab7 have been identified as a causative trigger for Charcot‐Marie‐Tooth Type 2B disease (CMT2B), inherited neuropathies affecting the peripheral nerves. Thus, CMT2B neuropathies are characterized by sensory loss, muscle weakness often leading to infections and consequently amputations. Disease causing mutations in Rab7 alter amino acid residues in the GTP‐binding and hydrolysis domains of Rab7. Through biochemical and morphologic assays we find the mutant GTPases exhibit altered functionality due to disrupted protein‐protein interactions and growth factor signaling. CMT2B mutants also exhibit increased GTP‐binding, and slowed membrane cycling, parameters that depend on regulatory guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP) interactions. We have established that Vps39 functions as a Rab7 GEF and TBC domain family, member 15 (TBC1D15) is reported to serve as a Rab7 GAP. Therefore, we comparatively evaluated wild‐type Rab7 and the four known CMT2B mutants (L129F, K157N, N161T, and V162M) in GEF and GAP assays. Regulatory protein facilitated nucleotide exchange and hydrolysis by N161T was comparable to Rab7 wild‐type. The remaining mutants were comparable to the constitutively active Rab7Q67L mutant. Thus, CMT2B neuropathy results from an interference with both Rab7 function and nucleotide activation status. NSF MCB‐0446179 (AWN) and NIH 2R25GM075149‐04 (PJ) grants support work.
Ligand design for human nicotinic acetylcholine receptors using in situ freeze‐frame click chemistry
Authors: Yamauchi John , Nemecz Akos , Ho Kwok‐Yiu , Fotsing Joseph R. , Weide Timo , Grimster Neil , Stump Bernhard , Talley Todd T. , Fokin Valery V. , Sharpless K. Barry , Taylor Palmer
Published: June 2021

Nicotinic acetylcholine receptors (nAChRs) are ionotropic receptors activated by acetylcholine in synapses of the CNS, neuromuscular junctions, and autonomic ganglia. The α4β2 and α7 nAChR subtypes are therapeutic targets for smoking cessation and treatment of schizophrenia. We developed a structure‐guided design strategy utilizing the Aplysia californica and Lymnaea stagnalis acetylcholine binding proteins (AChBPs) as homologous structural surrogates for the nicotinic receptor extracellular domain. We show that in situ click chemistry can be performed at subunit interfaces to generate selective nAChR agonists and antagonists. Lead compounds can then be refined through catalytic synthesis of close triazole congeners on a submilligram scale to assay AChBP template affinity and pharmacological activity on α4β2 and α7 nAChRs. Responses to other receptors that are potential complementary or “off targets” for the compounds were measured including α1 nAChR, 5‐HT3A and M1 muscarinic receptors. Crystallography of AChBP with the compounds was implemented to identify determinants involved in agonist or antagonist responses. The results of the functional and binding responses suggest that in situ freeze‐frame click chemistry can be a powerful tool in the future for developing specific ligands of various receptors. (Support from R37‐GM18360, UO1‐DA019372, NSF GK12 0742551)
Efficiency of sodium transport in the thick ascending limb
Authors: Nieves‐Gonzalez Aniel , Moore Leon C , Clausen Chris , Marcano Mariano , Layton Harold E , Layton Anita T
Published: June 2021

Thick ascending limb (TAL) cells are thought to transport Na+ efficiently owing to passive paracellular Na+ absorption. Transport efficiency is of particular importance in the outer medulla where O2 availability is limited by low blood flow. We used a mathematical model of TAL cells to estimate the efficiency of Na+ transport along the TAL, and to examine what determines transport efficiency. The TAL cell model includes all major solutes and transporters and channels, and is based on mass conservation and electroneutrality constraints. We analyzed TAL transport in cells with conditions relevant to the inner stripe of the outer medulla, the corticomedullary junction, and in the distal cortical TAL. At each location, we varied luminal [Na+] and [Cl−] and simulated a stop flow condition, where the luminal solute concentrations decrease to limiting, static‐head values. Na+ transport efficiency was calculated as the ratio of total net Na+ transport to transcellular Na+ transport. The results show that transport efficiency decreases as the transepithelial Na+ gradient increases. Based on predictions from countercurrent models, transport efficiency is predicted to be highest at the corticomedullary boundary where the transepithelial Na+ gradient is the smallest. As transepithelial Na+ gradient increases towards the deep inner stripe, transport efficiency decreases. Transport efficiency also decreases in the cortex as the luminal ion concentrations approach static head. Funded by NIH Grants SC1GM084744 and DK42091 and NSF grant DMS‐0715021
Inhibition of tyramine signaling by cGMP in Drosophila Malpighian tubules
Authors: Ruka Kristen A. , Blumenthal Edward M.
Published: June 2021

The Malpighian tubules are secretory epithelia that serve as the proximal component of the insect excretory system. The biogenic amine tyramine (TA) acts as a diuretic in Drosophila tubules by increasing transepithelial chloride conductance through a mechanism that requires an increase in intracellular calcium levels. The second messenger cGMP has previously been shown to cause diuresis by enhancing active transepithelial cation transport (Kerr et al., Curr. Biol., 2004). We tested the hypothesis that cGMP also affects the TA signaling pathway. Treatment of tubules with 1mM cGMP for 4 to 5 minutes resulted in a significant inhibition of the depolarizing response to both 10nM TA (80% suppression) and 100nM TA (18% suppression). Sensitivity to the diuretic peptide leucokinin (LK), which acts similarly to TA, was also inhibited by cGMP (38% suppression of response to 100pM LK). These data reveal the presence of some mechanism for cGMP‐mediated inhibition of diuretic signaling that is downstream of the TA receptor. We are currently investigating the involvement of specific cGMP‐dependent protein kinase isoforms in this modulation. Supported by NSF 0744619 to E.M.B.
Mechanisms of impaired vasopressin response in acute alcohol‐intoxicated hemorrhaged rats
Authors: Whitaker Annie M , Sulzer Jesse , Porter Johnny , Molina Patricia
Published: June 2021

Acute alcohol intoxication (AAI) blunts the neuroendocrine (catecholamine and arginine vasopressin (AVP)) response to hemorrhagic shock (HS) resulting in impaired blood pressure recovery during fluid resuscitation. Central stimulation of cholinergic nicotinic receptors restores AVP responses in AAI+HS suggesting that AAI disrupts signaling mechanisms controlling AVP release. We hypothesize the AAI‐mediated inhibition of supraoptic (SON) and paraventricular (PVN) responsiveness is controlled by alterations in the balance between inhibitory nitric oxide (NO) and stimulatory angiotensin II (ANG II) signaling. Chronically‐catheterized, adult male Sprague‐Dawley rats (250–300g) received a primed‐constant (2.5g/kg + 0.3g/kg/h) 15h intragastric infusion of alcohol or isocaloric/isovolumic dextrose (DEX). AAI produced a significant (~30%) increase in NO levels in the PVN and SON and NO synthase activity in the SON as compared to DEX controls. AAI did not alter circulating ANG II, but showed significant attenuation of ANG II in response to HS. These results suggest that the mechanisms underlying the attenuation of AVP release during HS in AAI involve altered balance between NO and ANG II control of hypothalamic responses. We predict that central inhibition of NO or ANG II stimulation will restore AVP and will in turn improve hemodynamic recovery from HS in AAI. Supported by DOD PR‐054196 and NIAAA‐AA7577.
Providing Research Experiences for Undergraduates (REU) from Rural & Tribal Colleges
Authors: Doze Van , Cisek Karen , Pyle Sally , Bennett T.M. Bull
Published: June 2021

For rural and tribal college students, a demographic underrepresented in research labs and graduate school programs, research opportunities at home institutions may be sparse. Undergraduate years are critical for engaging students in research to increase the number pursuing graduate work and careers in science. Recruiting and retaining students from these backgrounds, who may be hesitant to pursue opportunities based on social factors (e.g., first generation, non‐traditional, single parents), poses an added challenge. To reach these populations, we give special consideration to recruitment, lab experience, and retention. We partner with Native American scientists and faculty, conduct multiple campus visits for recruitment, and use peer mentoring and strategic pairing of new students with experienced students from similar backgrounds. The program's goals are to promote an interest in research and science careers, foster academic and social independence, and provide professional development. In addition to significant time receiving basic lab training and developing research projects, students attend professional development sessions. We have successfully trained >50 REU students. This has resulted in a significant number of rural and tribal students completing their undergraduate degrees and progressing to post‐baccalaureate programs. Supported by NIH P20RR0167141, NSF 0347259 and 0851869.
Construction and Validation of a DNA Combinatorial Library of 2E8 Combinations Designed for Solving Satisfiability Problems in Biomolecular Computing
Authors: Waters Colin , Bellomo Lea , Pogozelski Wendy
Published: June 2021

DNA is being investigated as a tool for biomolecular computing. Some of these biomolecular computing methods require the creation of combinatorial libraries. We have developed a method for creating combinatorial libraries of nearly any size. We demonstrate creation of library of 28 or 256 distinct DNA strands of 128bp, made by linking eight 16‐mer oligomers together. Each 16‐base oligomer has one of two possible sequences. One sequence is designated as “true” and the other as “false”. In computing terms, each library strand has eight variables with values of true or false (corresponding to 1 or 0 in a binary system). Every possible combination of the eight linked true or false strands (variables) yields 28 or 256 distinct library strands. The library was constructed by a series of primer extension and ligation steps. We used a variety of PCR‐based methods and gel electrophoresis to demonstrate that the sequences in the library have the expected length and composition.

This work was funded by NSF, the Camille and Henry Dreyfus Foundation, the Geneseo Student Association and the Geneseo Association.
Mathematical modeling of the gene regulatory network controlling the cold shock response in Saccharomyces cerevisiae
Authors: Vega Alondra J , Kuelbs Stephanie D , Fitzpatrick Ben G , Dahlquist Kam D
Published: June 2021

Transcription factors regulate gene expression by increasing or decreasing the rate at which a cell transcribes a gene. We are using mathematical modeling to describe the interactions between transcription factors thought to play a role in controlling the environmental stress response to cold shock in Saccharomyces cerevisiae. We created an initial network of 15 transcription factors that regulate one another. The concentrations of the individual transcription factors were modeled using ordinary differential equations, which we solved as a system in order to determine the behavior of the model. Each equation includes a production rate, a degradation rate (which we computed using published half‐life data), and weights that denote the influence of the connected transcription factors. We determined parameter values using DNA microarray data from the literature as well as from our laboratory. Model/data comparisons were performed with least squares. Model predictions fit the data well within two standard deviations of the experimental error. We further refined the model by adding and removing transcription factors in the network to examine their impact on regulatory behavior. This work was supported by NSF awards 0634613 (S.D.K., B.G.F., K.D.D.) and 0921038 (A.J.V., B.G.F., K.D.D.).
Promoter escape by E. coli RNA polymerase: its dependence on upstream rewinding potential of the −10/discriminator (DIS) region
Authors: Tabassum Doris Priscilla , Hsu Lilian M
Published: June 2021

Promoter escape governs the transition of RNA polymerase (RNAP) from the initiation phase to elongation. Being rate‐limited at this step, T5 phage N25 promoter undergoes high degree of abortive cycling prior to escape. Escape occurs at different positions depending on the initial transcribed sequence (Hsu et al. 2006 Biochemistry 45, 8841). As transcription proceeds, DNA scrunching (Revyakin et al. 2006 Science 314, 1139) leads to transcription bubble expansion to create a strained initial transcribing complex, which can result in successful escape or abortive release depending on which end of the expanded bubble rewinds. We hypothesized that changes in the upstream (−10/DIS region) DNA rewinding potential will affect the escape position of the N25 promoters. To alter the upstream rewinding potential, we introduced mismatched stretches on the template strand of the promoter while maintaining the contacts between the non‐template strand and the RNAP. We analyzed the constructs by in vitro transcription with [γ‐32P]‐ATP label followed by denaturing PAGE and phosphorimaging. Our results show that escape profiles are affected only when the mismatched regions are 5 bp or larger. We conclude that the upstream barrier to escape is imposed foremost by the strength of the σ2/−10 contacts and does not rely simply on the DNA‐composition dependent rewinding potential. Supported by NSF grant (RUI‐0841452) to L. M. Hsu
Testing the mechanism of very long abortive transcript (VLAT) formation via hyper forward translocation by E. coli RNA polymerase
Authors: Vallery Tenaya Kristine , Jiang Yunnan , Hsu Lilian M
Published: June 2021

Transcription initiation at the T5 phage N25 – an E coli Eσ70 – promoter is rate‐limited at the promoter escape step; this leads to the generation of an abortive RNA ladder of 11 nt with escape at +12. The length of the abortive ladder depends on the initial transcribed sequence (ITS). Thus N25anti, with ITS changes from +3 to +20, and DG203, with ITS changes from +3 to +10, abort to +15 and +19, respectively (Hsu et al. 2006 Biochemistry 45, 8841). Abortive RNAs ≤15 nt were shown to result from RNA polymerase (RNAP) backtracking, since their levels are ameliorated by GreB. In contrast, VLATs, referring to abortive RNAs 16–19 nt in length, are resistant to GreB and were deemed to result from RNAP hyper translocation (Chander et al. 2007 Biochemistry 46, 12687), during which RNAP forward translocates by >1 nt, thus overshooting the 3′‐end of the transcript. VLATs cannot be elongated further and are subsequently released from the arrested complex. To investigate the proposed VLAT mechanism, we placed an E111Q EcoRI roadblock at several locations in the DG203 downstream DNA, which bracket the promoter escape transition, to prevent forward translocation. Preliminary results reveal that E111Q EcoRI binds downstream DNA tightly to prevent VLAT formation in a predicted manner, thereby supporting the hyper forward translocation model. Supported by NSF Grant (RUI‐0841452) to L. M. Hsu
Identifying Novel Factors Involved In Yeast Mitochondrial Protein Synthesis
Authors: Gonzales Paul , Aguilar Mitzi , Tibbetts Anne S. , Appling Dean R.
Published: June 2021

The goal of our work is to identify protein factors involved in the initiation of mitochondrial translation using yeast genetics. FMT1 encodes the S. cerevisiae methionyl‐tRNA formyltransferase, which catalyzes the formylation of mitochondrial initiator Met‐tRNA. A genetic screen was performed with an fmt1 deletion strain to generate synthetic petite strains – strains containing mutations that in combination with the fmt1 deletion lead to the loss of respiratory growth. One mutant strain, s.p. 72–1, was transformed with a yeast genomic DNA library to identify open reading frames that complement the respiratory‐deficient phenotype. A region of chromosome XVI, including the LPE10 gene, was found to rescue growth on non‐fermentable carbon sources. Sequencing of DNA from the mutant strain revealed that the LPE10 gene, encoding a mitochondrial inner membrane magnesium transporter, contained a point mutation leading to a premature stop codon. This mutation likely causes the synthetic petite phenotype with the deletion of fmt1. Indeed, an lpe10 fmt1 double mutant has a more severe respiratory growth defect than the single lpe10 deletion strain. Deletion of the MIS1 gene, also required for the formylation of mitochondrial initiator tRNA, also leads to the petite phenotype in combination with the lpe10 deletion. This work is supported by HHMI and NSF grants to the Freshman Research Initiative at UT‐Austin.
Enhancing iron bioavailability of maize using a Caco2 cell/quantitative trait loci model
Authors: Lung'aho Mercy , Hoekenga Owen , Tako Elad , Glahn Raymond
Published: June 2021

Iron deficiency is an important public health problem that is estimated to affect over one‐half the world population. Improving the nutritional quality of staple food crops such as maize, by developing varieties with high bioavailable iron represents a sustainable and cost effective approach to alleviating iron malnutrition. The aim of our study was to use a Caco‐2 cell/quantitative trait loci (QTL) model to discover the genes that influence maize grain iron concentration and amount of bioavailable iron. After several breeding cycles guided by measurements of Fe bioavailability with the Caco‐2 cell model, we identified two maize lines with identical grain iron concentration (20 ppm), but one line contained double the amount of bioavailable iron (p < 0.001). Three QTL were found to influence the bioavailability of iron in the two maize lines. Blood hemoglobin data from a poultry model confirmed the results of the Caco‐2 cell model. Some of the lines screened using the Caco‐2 cell/QTL model have been grown at multiple locations and data show a genotype × environment (G×E) interaction. We conclude that conventional breeding can significantly improve iron bioavailability in maize grain and that the G×E effect plays a major role in Fe bioavailability. Human feeding trials should be conducted to determine the efficacyof consuming the high bioavailability maize.

Source of research support: United States Department of Agriculture, Cornell University
Iron status and serine hydroxymethyltransferase (SHMT) activity and abundance in pre‐ and postmenopausal women
Authors: Reddy Manju B. , Zhou Ying , Chen Dan , Alekel D. Lee , Hansen Christine M.
Published: June 2021

To explore relationships between iron status and lymphocyte SHMT activity (ACT) and abundance (ABN), 118 postmenopausal women (54.3 y) enrolled in SIRBL (Soy Isoflavones for Reducing Bone Loss) study and 105 young women (21.2 y) were studied. Serum ferritin, folate, and vitamin B‐12, plasma total homocysteine (tHcy) and pyridoxal phosphate (PLP), lymphocyte SHMT ACT and ABN were determined. Subjects in the first (4.8 ng/mL; 93% premenopausal) and fifth (92.6 ng/mL; 98% postmenopausal) quintiles of serum ferritin were compared. The fifth quintile had higher median BMI (25 vs 22 kg/m2; p<0.02), plasma PLP (p<0.0001), total homocysteine (p<0.05), and SHMT ABN (p<0.0001), but significantly lower SHMT ACT (p<0.02) than first quintile.

Ferritin quintile
Folate ng/mL
B‐12 pg/mL
PLP nmol/L
tHcy nmol/L
SHMT ACT cpm/ug protein
SHMT ABN relative density

First (n)
14.2 (45)
597 (45)
61.7 (45)
6.4 (45)
43.6 (27)
1.1 (21)

Fifth (n)
13.1 (45)
521 (45)
93.9 (45)
6.6 (45)
13.0 (14)
12.3 (31)

Increased iron status may enhance SHMT expression, favoring thymidine synthesis over Hcy remethylation, thus warranting further investigation.

Funded by USDA Special Grant, NWRC, Iowa State University.
Effective Bioactive Treatment for Ewing Sarcoma
Authors: Randolph Priscilla , Ibrahim Salam A. , Holmes‐McNary Minnie
Published: June 2021

Ewing sarcoma (EWS) is a rare form of bone cancer that is found primarily in Caucasian children and young adults. Ewing sarcoma is characterized as a highly malignant tumor that is found in both bone and soft tissues. However, when EWS is detected early the overall survival rate is 60%. Current treatment has been limited to chemotherapy, radiation and surgery or a combination of these treatment modalities.

Previous research by our lab and others demonstrate the beneficial cancer prevention properties of trans‐Resveratrol (RES) a bioactive nutrient. These former studies are the impetus for our present studies. Therefore, we hypothesize that RES will be an effective combinatorial treatment for EWS.

Our studies with human‐derived EWS cells show significant apoptotic cell death and differential gene expression during exposure with either TNF‐alpha, RES, irradiation (IRR) alone or in combination. Our studies clearly demonstrate an effective alternative treatment modality for EWS. Supported in part by NSF‐AGEP (PR), United States Department of Agriculture (USDA‐SI, PR), Faculty Initiated Grant (MHM), College of Arts and Sciences.

Grant Funding Source: NCAT, USDA
Broccoli alters estrogen metabolism in favor of 2‐methoxyestradiol in vitro
Authors: Volker Sonja E , Jeffery Elizabeth H
Published: June 2021

An increased 2‐hydroxy:16alpha‐hydroxy estrogen ratio may be correlated with reduced breast cancer risk. Estrogens are hydroxylated at the C‐2 position by cytochrome P450 1A (CYP1A). 2‐Hydroxyestrogens can be methylated by catechol‐O‐methyltransferase to form antitumorigenic 2‐methoxyestrogens, or can be oxidized to quinones that may react with DNA. Quinones may be reduced back to 2‐hydroxyestrogens by NAD(P)H:quinone oxidoreductase (NQO1). Broccoli contains glucosinolates that, upon hydrolysis, form bioactive isothiocyanates, which are known inducers of CYP1A and NQO1. Aqueous extracts of freeze‐dried broccoli powders were generated through overnight hydrolysis at room temperature (50 mg/mL water). Incubation of mouse hepatoma Hepa‐1c1c7 cells with broccoli extracts for 24 h induced CYP1A and NQO1 activities up to 72 and 4.9‐fold, respectively. When Hepa‐1c1c7 cells were incubated with 10 μM estradiol in the absence of broccoli extracts, no 2‐methoxyestradiol formation was observed by HPLC‐MS/MS. In contrast, a 24 h exposure of cells to broccoli extracts, followed by 24 h simultaneous exposure to broccoli extracts and 10 μM estradiol lead to formation of measurable amounts of 2‐methoxyestradiol in the culture medium. These results indicate that broccoli may redirect estrogen metabolism toward formation of anti‐tumorigenic 2‐methoxyestradiol, through induction of CYP1A and NQO1 activity. (supported by grants from USDA and NIH)
Effects of soy with high isoflavones vs casein protein diet and obesity on plasma isoflavones levels using obese DMBA‐induced mammary tumor Zucker rat model
Authors: Hakkak Reza , Shaaf Saeid , Talley Lori , Korourian Soheila
Published: June 2021

We reported that intact obese Zucker female rats fed semi‐purified diets were protected against DMBA‐induced mammary tumor on the casein but not soy diet. We investigate the effects of long‐term soy vs casein protein intake and obesity on plasma isoflavones levels. Five‐week‐old female Zucker rats were used. After one week of acclimation (age 42 days), rats were randomly assigned to; 1) Lean, casein; 2) Obese, casein; 3) Lean, soy and 4) Obese, soy. Rats had ad lib access to water and diet with either casein or soy protein isolate. Rats were orally gavaged at age 50 days with 65 mg/kg DMBA and sacrificed 155 days later. Plasma were collected to measure genistein, daidzein, glycitein, equol and O‐Desmethylangolensin (O‐MDA) (n=5/group). At end of the experiment, 69% of the Lean casein rats developed mammary tumors compared to 50% in lean soy group (p= 0.176). In the obese group, 76% of the soy‐fed rats developed mammary tumors compared to 15% (P<0.001) of obese casein‐ fed rats. Plasma daidzein and genistein concentration were not significantly different in lean vs obese soy‐fed rats. However, O‐MDA and equol concentration were significantly lower in obese soy‐fed rats compared to lean soy rats. These data suggests that obese and lean Zucker rats have different intestinal flora which may impact DMBA‐induced mammary tumor development. (Supported by USDA and ABI to RH)
Effects on mood and satiety of 4 days of partial energy deficit (60%) or energy excess (150%)
Authors: McGraw Susan M. , DeGroot David W , Ely Matthew R , Karl James P , Young Andrew J , Lieberman Harris R
Published: June 2021

We previously demonstrated that up to 48‐h of near total calorie deprivation does not affect mood or cognitive performance but does alter satiety. In this study we examined effects of partial energy deficiency (ED) and overfeeding (OF) on mood and satiety. Volunteers (10M, 4F, age 22 ± 2 yr, BMI 25.0 ± 3.8) underwent 4 days of EB, 4 days of ED (60% of energy balance (EB)), and 4 days of OF (150% of EB) in the laboratory. Macronutrient composition, daily activities and total energy expenditure were held constant. ED reduced self‐reported vigor compared to EB (p=0.02) as assessed by the Profile of Mood (POMS) state but OF had no effect on mood (Table). Both ED and OF affected perception of satiety as assessed with the SLIM questionnaire, with OF increasing satiety and ED decreasing it (p<0.001). This study indicates substantial, sustained underfeeding adversely affects mood but overfeeding does not, and that OF and ED both alter perception of hunger.

Mean POMS scores.

T
D
A
V
F
C
TOT

EB
2.98 ±3.1
1.24 ±2.2
1.86 ±2.3
16.7 ±8.6
2.88 ±4.7
3.24 ±2.2
−4.52 ±17.0

ED
3.67 ±2.3
1.57 ±2.1
2.02 ±2.8
12.8 ±9.0*
2.95 ±3.4
3.95 ±2.4
1.38 ±14.8

OF
2.60 ±2.0
0.67 ±1.1
0.81 ±1.6
13.76 ±9.5
3.43 ±4.1
3.74 ±2.4
−2.52 ±14.3

T, tension‐anxiety; D, depression‐dejection; A, anger‐hostility; V, vigor; F, fatigue; C, confusion;

TOT, Total mood score.

= Significantly different P=0.02 than EB

Funded by MRMC
Examination of fruit and vegetable preferences of low‐income public elementary school children
Authors: Wong Wei‐Ping , Lakkakula Anantha , Zanovec Michael , Tuuri Georgianna
Published: June 2021

High consumption of fruits and vegetables has established health benefits for children. Low income children may have reduced preferences for these nutrient‐dense foods due to lack of access to and familiarity with these items. The objective of this study was to examine fruit and vegetable preferences among low‐income elementary school children. A total of 280 third and fifth grade children (52% boys, 53% fifth graders) attending public schools in southeastern Louisiana participated. Children were shown pictures of 19 fruits and 19 vegetables and asked their opinion of each item. Apples, oranges, grapes, and bananas were the most liked fruits, and french‐fries, corn, green beans, and baked potatoes were the most liked vegetables. Papayas, avocados, apricots, mangos and kiwi were the least liked fruits, and coleslaw, cauliflower, onions, garlic, and spinach were the least liked vegetables. A factor analysis indicated that children's fruit and vegetable preferences grouped into 7 factors which explained 50% of the variance. All of the highly liked fruits grouped into the same factor while the most preferred vegetables were found in three different factors. Similarly the least liked fruits were found in the same factor while the least liked vegetables were found in four different factors. Children's preferences for fruit appear to be more consistent than that for vegetables.

Funding: USDA Grant #2009‐35200‐05287