bluuebunny/crossref_metadata_2025 huggingface dataset:
Relating Intein Flexibility to the Temperature Dependence of Activity
Authors: Yakely Anthony E. , Bonano Alexander A. , Jaramillo Mario V. , Williams Jennie E. , Reitter Julie N. , Mills Kenneth V.
Published: June 2021

Protein splicing is the self‐catalyzed removal of an intein, or intervening protein, in coordination with the ligation of the exteins, the flanking polypeptides. We are examining the DNA Pol II inteins from the extremophiles Pyrococcus abyssi and Pyrococcus horikoshii. The inteins from P. abyssi and P. horikoshii are largely homologous except for the presence of a large, disordered loop in the P. abyssi intein, in contrast to a smaller loop in the P. horikoshii intein. We have observed temperature dependence of the activity of the two inteins, which may be due to the difference in their structures. The smaller loop may allow for a more rigid structure in the P. horikoshii intein resulting in a higher temperature needed for protein splicing. The P. abyssi intein has a longer loop resulting in a less rigid structure, so a lower temperature is needed for protein splicing. To test this hypothesis, we used recombinant DNA to make a chimera of the P. abyssi intein with the P. horikoshii loop, and vice versa, to determine whether the change in loop alters the temperature dependence of the inteins’ activity. We successfully created the chimeric genes for both chimeric inteins and discovered that the proteins expressed well and were capable of splicing. For the P. abyssi intein with the P. horokoshii loop, we found that the chimeric intein spliced more than the native intein at a lower temperature but less at a higher temperature. For the P. horokoshii intein with the P. abyssi loop, we found that it displayed less splicing than the original protein at various temperatures. Comparative activity assays will be run to examine the temperature dependence of activity among the native and chimeric inteins.

Support or Funding Information

This material is based upon work supported by the National Science Foundation under grants MCB‐1244089 and MCB‐1517138 and by a Henry Dreyfus Teacher‐Scholar Award (KVM).
Influence of Intein Flexibility on Temperature Dependence of Protein Splicing
Authors: Long Joshua D. , Pierre Isabelle , Reitter Julie , Mills Kenneth V.
Published: June 2021

Protein splicing is a post‐translational modification by which intervening polypeptides called inteins catalyze their own excision from two flanking polypeptides, the exteins, concomitant with the ligation of the exteins. We studied the activity of DNA Polymerase II (PolII) inteins from the deep‐sea thermophiles Thermococcus kodakarensis (Tko), Thermococcus barophilus (Tba), Thermococcus sibiricus (Tsib), and Methanococcus infernus (Minf). Tko and Tba PolII inteins contain a homing endonuclease domain, while a flexible loop replaces this region in the Tsib and Minf inteins. This flexible loop, also found in Pyrococcus abyssi (Pab) and Pyrococcus horikoshii (Pho) PolII inteins, was hypothesized to be linked to the temperature dependent activity of these inteins. However, unlike Pab and Pho PolII inteins, we have shown that protein splicing and cleavage side reactions in the Tsib and Minf inteins occur in vivo in E. coli without the need for high temperature incubation in vitro. On the other hand, the Tba and Tko inteins splice poorly in E. coli and can be induced to splice in vitro at higher temperatures. We also have demonstrated that the Tba intein splices while the Tko intein undergoes a mixture of splicing and N‐terminal cleavage.

Support or Funding Information

This material is based upon work supported by the National Science Foundation under grants MCB‐0950245, MCB‐1244089 and MCB‐1517138 and by a Henry Dreyfus Teacher‐Scholar Award (KVM).
An Investigation of Salt‐Dependent Protein Splicing
Authors: Cousin Christopher E. , Reitter Julie N. , Nicastri Michael , Jaramillo Mario S. , Mills Kenneth V.
Published: June 2021

Protein splicing is a post‐translational modification where an internal polypeptide, called an intein, catalyzes its excision from two flanking polypeptides, called exteins, concomitant with extein ligation. Usually, when intein fusion proteins from other organisms are overexpressed in E. coli, splicing is spontaneous in vivo. However, despite a very similar sequence to the Mma PolII intein, which is capable of splicing, the Halobacterium salinarum (Hsa) PolII intein did not splice upon in vivo expression in E. coli. Halobacterium salinarum is a halophilic organism, so it lives in an environment with extremely high salt concentrations. In an effort to induce reactivity, two fusion proteins of the Hsa intein were incubated at 28°C for 16 hours with increasing amounts of NaCl. The fusion protein with a wild type intein has glutamine as the last residue of the intein, whereas the second intein, termed MIHQN, replaces this glutamine with asparagine. After incubation, in vitro splicing was observed in both intein fusion proteins on incubation with 2 M and 2.5 M NaCl.

Support or Funding Information

This material is based upon work supported by the National Science Foundation under grants MCB‐1244089 and MCB‐1517138 (KVM), by a Henry Dreyfus Teacher‐Scholar Award (KVM), and the Summer Research Fellowships from Renee and Anthony M. Marlon, M.D (CC).
9(Z),12(Z)‐Eicosadienoic Acid as a Probe for Substrate Binding by Soybean Lipoxygenase‐1
Authors: Hinnenkamp David J. , Clapp Charles H.
Published: June 2021

Soybean lipoxygenase is a non‐heme iron protein that catalyzes the oxygenation of linoleic acid and other polyunsaturated fatty acids to produce conjugated diene hydroperoxides. The manner in which substrates bind to this enzyme is uncertain. One hypothesis is that the terminal methyl group of substrates binds near phenylalanine‐557. To test this hypothesis, 9(Z),12(Z)‐eicosadienoic acid (9,12‐EDA) has been synthesized and studied as a substrate of wild‐type SBLO‐1 and the F557A mutant, in which the smaller residue at position 557 might allow accommodation of a fatty acid with a longer alkyl terminus. The kinetics are complicated by pronounced substrate inhibition at high concentrations of 9,12‐EDA. At low substrate concentration, 9,12‐EDA exhibits lower activity than linoleic acid with wild type SBLO‐1, but the activities of the two fatty acids are comparable with the F557A mutant. The results are consistent with the hypothesis that phe‐557 is located at the binding site for the methyl terminus of substrates.

Support or Funding Information

NSF CHE‐1213262
Concentration Effects in Identifying Unclumping Reagents
Authors: Zem Gregory , Mikaelian E. , Quinones L. , Nwokochah C. , Osadi F. , Arana D. , Barsigian S. , Dugyawi J. , Alvarado B. , Hovakemian G. , Lam J. , Eskander M. , Voldiner M. , Garcia S. , Nair D. , Song J. , Tashdjian G. , Nazer S. , Rashidizand S. , Constantino G. , Pilikian N. , Faretta A. , Gekchyan H. , Corscadden L. , Kelvani M , DeGuzman K. , Canta R. , Korkounian S. , Strelnicova A. , Turdjian M. , Lay M , Darmali A. , Memarian N , Simonyan K. , Oghlian L. , Oppenheimer S. B.
Published: June 2021

This study is part of a multi‐year effort to identify anti cell clumping reagents of potential usefulness in anti‐thrombocytic, anti‐cancer and anti‐infection applications. Here using a fixed (Prefer fixative, Anatech Ltd) yeast (Saccharomyces cerevisiae) model in deionized water, the effects of 3 concentrations (0.1M range) of 3 reagents (boric acid, polyethylene glycol and borax) were tested on their ability to unclump yeast as measured by percentage of single cells present over time (0 time, 20, 40, 60 min) versus controls in the absence of reagents. After 60 min based on 2700 experiments, the mean percent single cells compared with controls without reagents, for the 3 concentrations were: for boric acid +9, +16, +17; for polyethylene glycol +0.5, −2.5. −3.0; for borax −2.6, −0.7, +1.3. Boric acid was the only moderate unclumper identified here. In a typical set of 30 experiments with boric acid, the spread between control and experimental values increased substantially as time increased at reagent concentrations of 3, 6 and 9 mg reagent per ml of deionized water. By 60 min percent singles in experimentals and controls differed by about 70 percent with p values ranging from 0.0003 to 0.0000006 describing the significance of the spread. This study provides a precise evaluation of the effects of concentration in this model system suggesting that concentration may not substantially influence whether a reagent is an unclumper or not, under the conditions of the relatively high reagent concentrations and the specific protocol used here (support: Sidney Stern Memorial Trust, Cal State contributions from NSF Presidential Award 0731633 and various NIH and NSF grants).
Analysis of the Calyces from the Jamaica Species of Hibiscus Sabdariffa for Adrogenic Compounds
Authors: Butler Lakeisha Y , Morgan Melisa , Gray Wesley , Chin Kit , Snowden Janana
Published: June 2021

The leaves and flowers of Hibiscus sabdariffa are known to have many pharmacological properties. Hibiscus sabdariffa leaf extracts (HLE) have been documented to have anti‐cancer activities in different cancer cell lines. HLE has been shown to inhibit cell proliferation, migration, and metastasis in human prostate cancer cells suggesting that this flow may be a source for anti‐androgenic compounds. However, the anti‐androgen effect has not been examined in the calyx. The objective of this study was to obtain a unique bioactive fraction from the calyx of H. sabdariffa and to demonstrate its ability to inhibit prostate cancer cell growth. To accomplish our objective, we generated an aqueous and a methanol extraction of the calyces of the H. sabdariffa. The extracts were then examined by HPLC chromatography and for anti‐proliferative activity using the LNCaP cancer cells. Extraction of 22g of the calyces for 48hrs at room temperature resulted in 5.56mg and 2.75mg of analyte in aqueous and methanol extraction respectively. We obtained high yields of extracts (253mg per kg of dry weight) using water as compared to methanol as the solvent. An HPLC chromatogram profile of each extract was generated to characterize the number of respective chemicals present in the analytes. Analysis of the methanol extract revealed the presence of at least two distinct peaks with a retention times of 3.12min and 3.27min. We observed that the aqueous extract resulted only in a single peak with a retention time of 3.02min. Next, we examined the ability of both extracts to inhibit the growth of LNCaP‐GFP cancer cell line. Treatment of these cells for 1–5 days resulted in dose and time dependent inhibition of cell growth. We found that the methanol extract was capable of inducing cytotoxicity in 24hrs as compared to aqueous extracts which induced cytotoxicity after 72hrs. Our results demonstrated that both methanol and aqueous extracts of H. sabdariffa contained growth inhibitory compounds.

Support or Funding Information

NIH R15AT006821(WG) and USDA/NIFA 2012‐38821‐20092.
Biological Activity of Outer Membrane Vesicles from Clinical Isolates of Escherichia coli
Authors: Morales‐Lozada Yermary , Báez‐Bravo Gabriela , Ferrer‐Acosta Yancy , Gómez‐Moreno Ramón , Baerga‐Ortiz Abel
Published: June 2021

All gram‐negative bacteria secrete outer membrane vesicles (OMV) that have several functions, among them the transportation of bacterial virulence and survival factors. It has been found by other groups that certain OMV promote aberrant growth in mammalian cells upon contact. OMV provide an insoluble secretion pathway for gram‐negative bacteria and are responsible for the release of outer membrane and periplasm content out of the cell. The main objective of this investigation is to compare the composition and biological activity of OMV isolated from diverse clinical strains of E. coli. We will assess morphological effects of OMV from selected clinical isolates of E. coli on HeLa cells. Furthermore, we will also measure the lipid composition of OMV for the different strains and mutants by GC/MS. As preliminary results, we observed a significant difference in the lipid composition of the IHE3034 (pks+ strain) OMV and the IHE3034‐ΔclbP OMV. The clbP gene is presumed to be involved in the biosynthesis of colibactin. We observed that the deletion of clbP peptidase causes a decrease in the percentage of 12:0 fatty acids and an increase of 16:0 fatty acids in OMV, but not in the cellular membrane. These results begin to establish a correlation between the presence of colibactin genes and events at the bacterial cell surface.

Support or Funding Information

NSF grant CHE0953254 NIH grant R25GM061838 (MBRS‐RISE program) to Ramón Gómez‐Moreno
Crystallographic Study of Wild‐type and Mg2+‐free K‐Ras GTPase
Authors: Parker Jillian , Mattos Carla
Published: June 2021

The small GTPase Ras has been widely studied due to its relevance in cancer and other developmental disorders, termed RASopothies. When bound to GTP, Ras conveys signals to downstream effectors to control cellular signaling processes such as proliferation, differentiation, and apoptosis. Hydrolysis of GTP to GDP and inorganic phosphate (Pi) halts Ras signaling. This switching mechanism is tightly regulated by GEFs (guanine nucleotide exchange factors) and GAPs (GTPase activating proteins), where oncogenic mutations in Ras lead to uncontrolled Ras cell cycling and tumorigenesis. To date, structures of the most oncogenic isoform, K‐Ras, in its activated GTP‐bound form have been difficult to obtain, with only a couple of its mutants currently deposited in the Protein Data Bank (PDB). The first crystal structure of wild‐type K‐Ras bound to the GTP‐analogue GppCH2p has been obtained to 1.89Å resolution. This structure shows K‐Ras in the state 1 conformation, with switch I away from the active site without threonine 35 interacting with the Mg2+ or nucleotide. Additionally, structures of active K‐Ras obtained from lower pH conditions show perturbed active site elements, the most striking of which is the absence of the canonical Mg2+ ion in the nucleotide binding pocket. The Mg2+‐less structures exhibit both local and long‐range alterations in residues located within conserved nucleotide‐binding motifs and involved in sensing the state of the nucleotide bound in the active site. Additionally, similarities are seen between Mg2+‐less K‐Ras and nucleotide‐less Ras found in complex with Sos, the most common Ras GEF. These results shed light on why the active form of K‐Ras has been structurally difficult to characterize, as well as provide a structural basis for more focused targeting of the K‐Ras isoform.

Support or Funding Information

NSF MCB‐1244203
Allosteric role of R97 on the NRas active site
Authors: Reid Derion F , Mattos Carla
Published: June 2021

Ras proteins are small GTPases that are involved in signal transduction pathways that controls cellular proliferation, differentiation, and survival. Ras is constitutively activated in roughly 20% of human cancers. The Ras subfamily is made up of H‐, K‐, and NRas, where NRas is the least studied. NRas functions as a molecular switch that exchanges guanosine diphosphate (GDP) for guanosine triphosphate (GTP) rendering activation, and hydrolyzes GTP to GDP for deactivation. Once activated, NRas interacts with Raf, an effector molecule that our lab has hypothesized to allosterically modulate intrinsic hydrolysis. The catalytic G domain of NRas comprises an effector lobe (residues 1–86) and allosteric lobe (residues 87–166). In our allosteric model, switch II of Ras becomes ordered when a negatively charged ligand binds between helix 3 and loop 7 allowing for intrinsic hydrolysis. Molecular dynamics studies of Ras bound to Raf revealed R97 of helix 3 becomes less dynamic upon binding of Raf, coordinating the binding of a negatively charged ligand at the allosteric site. Interestingly, R97G is a rare mutation of NRas, found in a human tumor. Here, we present the structure of NRasR97G bound to the non‐hydrolysable GTP analogue, GppNHp. The structure and hydrolysis rates of NRasR97G are compared to those of wild‐type NRas.

Support or Funding Information

This research was funded by NSF Grant Number MCB‐1244203.
Mechanistic Implications of the Unique Structural Features of the Anti‐Sigma Domain of the Pseudomonas Sigma Regulator, PupR
Authors: Jensen Jaime , Balbo Andrea , Neau David , Chakravarthy Srinivas , Zhao Huaying , Sinha Sangita , Colbert Christopher
Published: June 2021

Gram‐negative bacteria tightly regulate intracellular iron, an essential nutrient. To ensure this strict control, some outer membrane TonB‐dependent transporters (TBDTs) responsible for iron import stimulate their own transcription in response to extracellular binding of ferric siderophore. This process is mediated by an inner membrane sigma regulator protein that transduces an unknown periplasmic signal from the TBDT to release an intracellular sigma factor from the inner membrane, which ultimately upregulates TBDT transcription. Here, we employ the Pseudomonas putida ferric‐pseudobactin BN7/BN8 sigma regulator, PupR, as a model system to understand the molecular mechanism of this conserved class of sigma regulators. We have determined the X‐ray crystal structure of the cytoplasmic anti‐sigma domain (ASD) of PupR to 2.0 Å. Size exclusion chromatography, small‐angle X‐ray scattering, and sedimentation velocity analytical ultracentrifugation (SV‐AUC) all indicate that, in contrast to other ASDs, the PupR‐ASD exists as a dimer in solution. Mutagenesis of residues at the dimer interface identified from the crystal structure disrupts dimerization and protein stability, as determined by SV‐AUC and thermal denaturation circular dichroism spectroscopy. Additionally, we have demonstrated that the helical content of the PupR‐ASD increases in the presence of trifluoroethanol, and are investigating the interaction of the PupR‐ASD with its cognate sigma factor, PupI. These combined results suggest that this type of inner membrane sigma regulator may utilize an unusual mechanism to sequester their cognate sigma factors and prevent transcriptional activation.

Support or Funding Information

Funded by NSF ND EPSCoR DDA #FAR0025216 to JJ; NIH NINDS 1R03 NS090939 and NSF MCB‐1413525 to SS; NIH NIGMS 1R15 GM113227, NIH NCRR 2P20 RR015566, and NIH NIGMS P30 GM103332 to CC.
Structural Biology of Aromatic Amino Acid Biosynthesis in Plants: The Prephenate Branch Point
Authors: Holland Cynthia K , Jez Joseph M
Published: June 2021

Plants are chemists that have spent the last 425 million years evolving unique secondary metabolites to communicate with their surroundings, defend themselves against pathogens, insects, and herbivores, and grow and develop. Many secondary metabolites are biosynthesized from the aromatic amino acids tryptophan, tyrosine, and phenylalanine, and the shikimate and aromatic amino acid biosynthetic pathways link primary and specialized metabolism. Prephenate aminotransferase (PAT) is a key enzyme in aromatic amino acid biosynthesis because its product, arogenate, is the immediate precursor for both tyrosine and phenylalanine. Interestingly, the enzyme has a dual role in metabolism and can also function as a classical aspartate aminotransferase. AtPAT is pyridoxal 5′‐phosphate‐dependent (PLP) enzyme belonging to the AAT‐like aspartate aminotransferase domain and follows a ping‐pong bi‐bi reaction mechanism through Schiff‐base formation to a catalytic K306 in the active site. Each subunit of the AtPAT dimer consists of a large domain and a small domain that belong to the α‐β class of protein fold. Despite the structural similarities to aspartate aminotransferases, the molecular basis of prephenate binding specificity remains elusive. To gain an understanding of the enzyme's chemistry and substrate selectivity, a 2.5 Å x‐ray structure of PAT from the model plant Arabidopsis thaliana (thale cress) was solved in complex with the PLP. To our knowledge, this is the first structure of a bona fide prephenate aminotransferase. Amino acid sequence comparisons of all known functional PATs shows that T84 and K169 are highly conserved; however, some enzymes annotated as PAT display sequence variations at these positions. Biochemical analysis of T84V, K169V, and T84V/K169V PAT reveals that the double mutant gained tyrosine aminotransferase activity and was able to convert 4‐hydroxyphenylpyruvate to tyrosine. T84 is on the opposite side of the active site pocket, while the amine backbone of K169 hydrogen bonds to PLP. We hypothesize that Thr84 and Lys169 are important for substrate recognition in the active site and will use these mutants in future crystallographic experiments. Overall, our structural studies will assist in understanding the role of this bi‐functional enzyme in the context of aromatic amino acid biosynthesis in plants.

Support or Funding Information

This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE‐1143954. Any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. Results shown in this report are derived from work performed at Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source. Argonne is operated by UChicago Argonne, LLC, for the U.S. Department of Energy, Office of Biological and Environmental Research under contract DE‐AC02‐06CH11357.
Failure to Achieve Recommended ω‐3 DHA Intake During Pregnancy: Nutrition Education Needed
Authors: Thaxton Carly A , Gaitan Adriana V , Drewery Merritt L , Pinkston Rakeysha I , Seidemann Ericka L , Elkind‐Hirsch Karen , Lammi‐Keefe Carol J
Published: June 2021

Dietary Guidelines for Americans 2010 recommends pregnant or breastfeeding women consume 8–12 oz of seafood/week, translating to an average DHA+EPA intake of 200–250 mg/day. In an on‐going study of overweight pregnant women in Louisiana, we assessed dietary intake and fatty acid status of red blood cells (RBC) mid‐pregnancy. Women (n=10, 20–34 years of age) entered the study at 17–20 weeks of pregnancy (age: n=5 ≥25 y, n=5 <25 y; ethnicity: n=5 African American, n=5 White; SES: n=5 low SES, n=5 med/high SES). Dietary intake was assessed by repeated (n=7) 24‐hour dietary recalls using the University of Minnesota Nutrient Data System for Research (NDSR). Red blood cell fatty acid concentrations were determined with gas chromatography at the time of study entrance. The relative weight percentage (wt%) of each RBC fatty acid identified was determined from the retention time. On average, women consumed 93±59 mg DHA+EPA/day via diet; n=1 achieved the recommended intake of 200 mg/day. Taking prenatal supplementation into consideration, the average DHA+EPA intake was 199±96 mg/day; n=5 achieved the recommended intake of 200 mg/day. While all enrolled women (n=10) consumed prenatal supplements during pregnancy, not all prenatal supplements contained DHA+EPA (n=1). On average, prenatal supplements provided an additional 121±77 mg DHA+EPA/day. Including prenatal supplement consumption, the average ω‐6:ω‐3 ratio was 1.01±0.78. There was a significant difference in DHA+EPA intake between the low SES and med/high SES women, (P=0.01); low SES had higher intake (255±70 mg/day) than med/high SES (115±78 mg/day). Ethnicity and age did not significantly affect DHA+EPA intake (P≥0.3). The top 5 dietary sources of DHA+EPA were crawfish, chicken, shrimp, eggs, and crab. Other seafood sources commonly consumed included catfish, oysters, and tilapia. In terms of fatty acid status, the wt% of RBC DHA was 9.25±1.3 (range: 7.33–11.05) and RBC EPA was 0.14±0.03 (range: 0.1–0.22). There were no significant differences in RBC DHA+EPA status for SES, age, or ethnicity (P≥0.2). Furthermore, there was no correlation between DHA+EPA intake and RBC concentrations (r=−0.06). The latter observation may be explained, in part, by the fact that DHA is preferentially transferred across the placenta to meet fetal demands during gestation. These data point to a need for nutrition education for pregnant women and women of childbearing ages regarding the importance of consuming fish varieties that are highly enriched in DHA+EPA.

Support or Funding Information

Funded in part by: LSU Undergraduate Research Grant (C Thaxton); USDA AFRI 2012‐67017‐19293 (CJL‐K); and LSU AgCenter.
The Effect Of Maternal Supplementation With A Lipid‐Based Nutrient Supplement On Infant Micronutrient Intake In Guatemalan Women And Infants
Authors: Haber Juliana A. , Solomons Noel W. , Hampel Daniela , Orozco Mónica N. , Allen Lindsay H.
Published: June 2021

BACKGROUND

According to the World Health Organization (WHO), infants should be exclusively breastfed for the first 6 months of life. Despite the high adherence to the WHO guidelines in Guatemala, there is a high prevalence of malnutrition during early life, which increases during the first 6 months of age. This suggests that breast milk quality may be suboptimal with regard to provision of micronutrients. Our objective was to evaluate whether maternal supplementation with a lipid based nutrient supplement (LNS) would increase infant micronutrient intake from breast milk.

METHODS

The randomly‐allocated, cross‐over design included 30 mother‐infant dyads 4–6 months postpartum in Quetzaltenango, Guatemala. In three 8‐hour milk collection visits the participating mother received a single bolus dose of 30 g LNS (B), 10 g of LNS divided over 3 time points (D), or no LNS (C). Mid‐feed milk samples were collected at each infant feed and total milk consumption measured. Maternal blood was collected at baseline, maternal dietary intake was assessed, and maternal and infant anthropometry was measured. The primary outcome was infant B‐complex vitamin intake from breast milk, estimated using micronutrient analyses and infant milk consumption. Analysis of variance and pair‐wise comparisons were used to examine the effect of dosing regimen. Clinicaltrials.gov (NCT02464111on).

FINDINGS

Of 26 dyads completing the study, results for 8 are included in this preliminary data analysis. On average, during 8 hours infants consumed more riboflavin when their mother received LNS (B or D), compared to C (9±6 μg, treatment p=0.012). B increased infant riboflavin intake by 28±21 μg, (p=0.005) whereas D increased intake by 19±14 μg (p=0.018). Neither B nor D increased infant pyridoxal (B 37±29 μg, D 44±36 μg, C 23±10 μg, p=0.328), niacin (B 41±31 μg D 37±30 μg, C 30±17 μg, p= 0.490) or thiamin (B 4±2 μg, D 6±6 μg, C 4±3 μg, p= 0.408) intake compared to no‐supplement. Compared to C, over 8 hours B provided an extra 6%, 0%, 14%, 1% of the Adequate Intake for infants under 6 months for riboflavin, thiamin, pyridoxal, and niacin, respectively, whereas D provided an extra 3%, 1%, 21%, 0%, respectively.

INTERPRETATION

Maternal supplementation with LNS as a bolus dose or divided dose increased infant riboflavin intake, with the bolus dose proving more efficacious at increasing infant riboflavin intake from milk. In this initial group of dyads, maternal supplementation with LNS did not significantly impact infant pyridoxal, niacin, or thiamin intake.

Support or Funding Information

UC Global Health Initiative, Blum Center for Developing Economies, UC Davis, USDA ARS Western Human Nutrition Research Center.
Cognitive Interviews with Parents to Validate a Questionnaire Assessing the Quality of Home Food Supplies
Authors: Delaney Colleen , McKinley Jessica , Martin‐Biggers Jennifer T , Byrd‐Bredbenner Carol
Published: June 2021

Foods available at home can affect health outcomes. Time‐efficient, reliable, valid tools that accurately estimate the calorie and nutrient quality of the home food supply are needed. Thus, a 36‐item food frequency questionnaire for individual intake was modified to assess household food supply fruit and vegetable servings, calories, percent calories from fat, and nutrients (i.e., total fat, saturated fat, cholesterol, vitamin C, fiber, potassium, magnesium, sugar from sweetened beverages). As part of validation, cognitive interviews were conducted with 15 parents to assess comprehension of questionnaire items and accuracy of interpretation by having parents read each questionnaire item aloud and verbally explain what the item was asking, then select their answer to the item and state the reasoning for the answer. The instructions for completing the questionnaire were reviewed with each parent before the cognitive interview began. After the first 5 interviews, the instrument layout was revised to put fewer items on each page (similar items, such as fruits and vegetables, were grouped together on a page) and repeating questionnaire instructions at the top of each page as a reminder of how to answer the items. Overall, parents correctly interpreted questionnaire items, felt that they were quick and easy to complete, and had only minor suggestions for improvement. A few parents thought some items were redundant, mainly because parents did not differentiate between food descriptors/qualifiers (e.g., low fat vs whole milk) indicating a need for stronger visual cues in the questionnaire to facilitate differentiation (e.g., color, bold, and/or underline descriptors). Several were unsure as to whether takeout food, specifically pizza, should be counted as part of the household food supply. A few wanted to report on additional food items beyond those in the questionnaire, such as ethnic foods. Some had minor difficulties remembering the questionnaire was about household food availability not dietary intake of individual family members. Cognitive interview results indicate that a food frequency questionnaire modified for assessing household food supply is a quick, easy tool for estimating food availability in the home and is understandable and acceptable to study participants. Future research should investigate the validity of the questionnaire by comparing parent responses to actual household food supplies.

Support or Funding Information

USDA NIFA #2011‐68001‐30170
Fruit and Vegetable Consumption and Physical Activity in Young Adults: Get Fruved Study
Authors: Barr Makenzie L , Olfert Melissa D , Zhou Wenjun , Riggsbee Kristin , Mathews Anne EW , Vilaro Melissa , Kattelmann Kendra K. , Leischner Krista , Crouter Scott , Morrell Jesse S , Thompson Dixie , White Adrienne , Horacek Tanya , Kidd Tandalayo , Brown Onikia , Colby Sarah E
Published: June 2021

The objective of this multi‐state research collaborative effort “Get Fruved” was to identify consumption of fruits and vegetables and physical activity across four university populations and geographical locations to compare for differences. All four university research teams enrolled freshmen, sophomore, and junior college students from Florida, South Dakota, Tennessee and West Virginia in two multi‐university training courses to develop a peer‐led, social marketing and environmental intervention in collaboration with researchers and Extension Professionals. Baseline assessments included a behavioral and demographical survey. Demographical variables included living on or off campus, relationship status, hours working a job, GPA, if they were a student athlete, scholarship status, had a Pell Grant or were vegetarian. Baseline demographics and geographical data were analyzed by FV consumption (NCI Fruit and Vegetables Screener) and physical activity (International Physical Activity Questionnaire) using one‐way ANOVA testing. The participants (N=280) were predominately Caucasian (84.40%), female (87.89%), and 18–20 years of age (75.29%). Year in school varied: sophomores (39.84%), juniors (34.38%), and freshmen (18.75%). Participants were primarily White (84.40%), with the remaining being Black or African American (5.05%), Hispanic or Latino (1.38%), and other (including biracial) (9.17%). Student athletes had higher levels of physical activity than non‐student athletes (p<0.0001; 4905.62 MET‐min/wk vs. 2729.98, respectively). Males were more physically active than females (p=0.0038; 3783.36 MET‐min/wk vs. 2712.88, respectively). Vegetarians had higher levels of FV consumption than non‐vegetarians (p=0.0027; 5.45 cups vs. 3.52 cups, respectively). No other differences were found in behavioral variables across demographical groups, including across geographical locations and across gender The findings indicated that the assessments used adequately captured characteristic and behavior relationships that would be expected based on previous research (e.g. vegetarians with increased FV consumption and student athletes with increased PA). Similar characteristics of participants across the four states for FV intake and PA provide a baseline for future intervention implementation.

Support or Funding Information

Funding support USDA/AFRI #2014‐67001‐21851
Sleep and Stress Level of College Students Developing a Health Promotion Intervention: Get Fruved Study
Authors: Olfert Melissa D. , Barr Makenzie L. , Zhou Wenjun , Riggsbee Kristin , Mathews Anne EW , Vilaro Melissa , Kattelmann Kendra K. , Andrea Hanson , Spurgeon Shawn , Morrell Jesse S , Franzen‐Castle Lisa , Byrd‐Bredbenner Carol , White Adrienne , Horacek Tanya , Kidd Tandalayo , Brown Onikia , Colby Sarah E
Published: June 2021

The objective of this study was to examine sleep hours (SH) and perceived stress (PS) levels in college students. Freshmen, sophomore, junior and senior college students (n=280) from four land‐grant, public universities (Florida, South Dakota, Tennessee and West Virginia) where enrolled in the “Get Fruved” research study and trained via two multi‐university courses. In the courses, the students worked with researchers and Extension Professionals to develop a peer‐led, social marketing and environmental change intervention at each of their respective campuses. Baseline assessments included behavioral (SH and PS) and demographic characteristics (including age, race/ethnicity, residing on/off campus, # hours working, relationship status, GPA, student athlete status, financial aid). SD was determined by the Pittsburgh Sleep Quality Index; PS via Cohen Perceived Stress Scale. Demographic differences in SD and PS were evaluated by one‐way ANOVA. SD (x=7.3±1.1) differed between universities (p<0.01), among age groups (p<0.05), and among different working hour groups (p<0.01). Hispanic students reported higher levels of stress compared to students not identifying as Hispanic (25.65 vs. 22.58, p<0.01); Asian students reported higher levels of stress than students of other races (27.53 vs. 22.63 p<0.01). SD and PS did not differ between students living on/off campus, by relationship status, GPA, athlete status, or financial aid status. Future wellness interventions targeting college students should consider geographical, racial/ethnic, and behavioral differences as they relate to sleep and perceived stress.

Support or Funding Information

Funding support USDA/AFRI #2014‐67001‐21851
Developing Food‐Specific Molecular Profiles and Biomarkers of Food and Nutrient Intake, and Dietary Exposure for Reducing Cancer Risks
Authors: Emenaker Nancy J. , Chester Deirdra N.
Published: June 2021

Background

Dietary and lifestyle choices comprise significant environmental modifiers contributing to disease risks, including diabetes, heart disease, and cancer. Yet few associational diet studies yield reliable health strategies reducing disease incidence. Reliable and quantitative methods are needed for measuring dietary consumption and compliance that advances our knowledge beyond association between dietary intake, health and disease etiology.

Objective

To describe food‐specific molecular profiles and biomarkers development of functional foods, nutrients and dietary exposures for reducing the risk of disease, in particular cancer.

Methods

Growing evidence indicates molecular markers or food‐specific signatures could provide potentially useful information about dietary consumption. For example, recent evidence demonstrated metabolic profiles correspond to high animal protein, or fruit and wine consumption highlighting the importance of metabolomics and stratification of individuals with respect to dietary habits.

Results

NIH and USDA‐NIFA jointly support and encourage research addressing the specific gaps on food specific molecular signatures and biomarkers of food and nutrient intake, and dietary exposure over time using both animal and human studies as appropriate. Clinical studies that involve controlled feeding and short term bolus feeding studies involving limited human subjects are appropriate for this purpose. Research approaches of interest include but are not limited to:

Identification and validation of food and nutrient specific metabolic signatures that correlate with nutrient quality and efficacy and nutrient consumption;

Identification and validation of molecular signatures of dietary consumption of nutrients over time, including commonly used nutrient supplements, and energy supplements or beverages;

Studies that explore the interaction/competition between various nutrients including natural products for their absorption, transport, metabolism and elimination;

Studies that explore the interaction/competition between various nutrients and drugs for their absorption, transport, metabolism and elimination, dose response, bioavailability, toxicity, and ADME profiles; and

Studies that explore natural products/nutrients, microbiota interactions with host physiology and metabolism.

Conclusion

Development of reliable quantitative molecular markers or food specific signatures may advance the field beyond associational links between dietary intake, health and disease etiology.

Support or Funding Information

Funding supported by the NCI/NIH and USDA.
Raisin Consumption is Associated with Increased Nutrient Intake, Better Diet Quality, and Lower Body Mass Index in Adult Participants (19+ years) from the NHANES (2001–2012)
Authors: Fulgoni Victor L , O'Neil Carol E , Painter James , Carughi Arianna
Published: June 2021

The association between raisin consumption with nutrient intake, dietary quality, and weight parameters was examined in adults using data from National Health and Nutrition Examination Survey (NHANES), 2001–2012 (n=29,684). Raisin consumers (n=458, 60.1% female) were defined as reporting consumption of any amount of raisins during the first 24‐hour diet recall. Diet quality was calculated using the Healthy Eating Index‐2010 (HEI). Covariate adjusted (age, gender, race/ethnicity, poverty income ratio, physical activity level, current smoking status, and alcohol intake) regression analyses were conducted using appropriate sample weights; significance was set at p<0.01. For analyses of nutrients and food groups energy intake was an additional covariate. Energy intake was higher in raisin consumers than non‐consumers (2257 ± 44 vs. 2074 ± 9). Examining nutrients of public health concern/shortfall nutrients identified by the Dietary Guidelines Advisory Committee Report 2015, dietary fiber (22.1 ± 1 vs. 16.5 ± 0.1 g/d) and potassium (3084 ± 77 vs. 2665 ± 10 mg/d) were significantly higher in raisin consumers compared to non‐consumers, while no associations were observed for intakes of calcium and vitamin D. Intakes of vitamin A, folate, and iron were similar in raisin consumers and non‐consumers but intake of vitamin C (117 ± 8 vs. 94.4 ± 1.2 mg/d), vitamin E (8.9 ± 0.5 vs. 7.3 ± 0.1 mg AT/d), and magnesium (355 ± 11 vs. 290 ± 1 mg/d) were higher in raisin consumers. Regarding nutrients to limit, intakes of saturated fatty acids (21. 2 ± 0.9 vs. 25.0 ± 0.1 g/day), added sugars (14.9 ± 0.7 vs. 18.0 ± 0.2 tsp equivalents/day), and sodium (3190 ± 78 vs. 3541 ± 17 mg/day) were significantly lower in the raisin consumers vs. non‐consumers. Consumers had higher total HEI scores (61.4 ± 1.0 vs. 49.1 ± 0.2). Regarding HEI sub‐components, except for total dairy and total protein, HEI sub‐component scores were higher for raisin consumers as compared to non‐consumers. The largest differences in sub‐component scores were seen for calories from solid fats, added sugars, and alcohol, (14.3 ± 0.4 vs. 12.1 ± 0.1), whole grains (4.2 ± 0.2 vs. 2.1 ± 0.04), and whole fruit (3.8 ± 0.2 vs. 2.1 ± 0.03). BMI (27.3±0.4 vs. 28.8±0.1 kg/m2) and waist circumference (94.1±0.1 vs. 97.8±0.2 cm) were lower in consumers. Raisin consumers were 39% less likely [odds ratio (OR):0.61, 99th confidence interval (CI): 0.41, 0.89] to be overweight or obese and had a 48% reduced risk (OR=0.58, 99th CI: 0.36, 0.74) of increased waist size. Raisin consumption was associated with better nutrient intake, diet quality and weight parameters in adults.

Support or Funding Information

Funding provided by Sun‐Maid Growers of California and USDA.
In vitro Short‐Chain Fatty Acid Production of Rice Bran Components by Human Gut Microbiota
Authors: Pham Tung , Savary Brett J , Chen Ming‐Hsuan , Lee Sun‐Ok
Published: June 2021

Rice bran is a rich source of bioactive components that have potentials to promote gastrointestinal health. However, bran is typically removed during polishing. Among those, feruloylated arabinoxylan oligosaccharides (FAXO) and rice bran polyphenolics (RBPP) are believed to have positive impacts on human gut microbiota. The objective of this study is to investigate the ability of FAXO and 50% ethanol fraction of RBPP to increase short‐chain fatty acids (SCFA) production, including acetic, propionic, and butyric acid, which are strongly associated with colonic health in human. Fresh fecal samples collected from five healthy adults with no signs or symptoms of bowel diseases or conditions were diluted with anaerobic medium. Each sample received 5 treatments separately: Blank (no substrates), Control (fructooligosaccharides – FOS, prebiotic), FAXO, RBPP, and FAXO with RBPP. Samples were prepared inside an anaerobic chamber, then capped and sealed tightly. Samples were incubated at 37°C. An aliquot of 1.5 mL was drawn from each treatment test tube at 0, 4, 8, 12, 24, and 30 hours and stored immediately at −80°C. SCFA concentrations were measured quantitatively using gas chromatography. Results showed that SCFA productions were increased with the addition of FAXO. Although total SCFA production of FAXO was not significantly higher than that of FOS, individual SCFA productions of FAXO were significantly higher compared to FOS (P<0.05). Propionic acid production of FAXO was higher compared to FOS at timepoint 24‐hour (2.32±0.70 mM and 0.34±0.36 mM, respectively) and 30‐hour (2.48±0.70 mM and 0.17±0.61 mM, respectively). FAXO also produced more butyric acid compared to FOS at 12‐hour (1.98±0.24 mM and 0.36±0.76 mM, respectively), 24‐hour (2.53±0.48 mM and 0.56±0.58 mM, respectively), and 30‐hour (2.90±0.80 mM and 0.51±0.62 mM, respectively). However, the synergistic effects of FAXO and RBPP were not observed as their combination did not increase SCFA productions significantly compared to FAXO treatment. Results from this study showed that FAXO exhibited prebiotic‐like activities and can potentially be used as a functional ingredient to promote colon health.

Support or Funding Information

Funding provided by the USDA NIFA (Grant No. 2014‐67017‐21766) is gratefully acknowledged.
Fructokinase prevents the hyperfructosemia of human hereditary fructosuria and the hyperglycemia of fructose‐induced metabolic syndrome
Authors: Ferraris Ronaldo P , Sugimoto Keiichiro , Patel Chirag , Douard Veronique , Shah Ami , Inui Hiroshi , Yamanouchi Toshikazu
Published: June 2021

We determined whether the effects of dietary fructose on blood fructose and glucose concentrations are modulated by fructokinase (FK) and the fructose transporter GLUT5. Portal and systemic fructose levels determined by HPLC in wildtype (WT) mice and rats fed for 5 d 0% free fructose were <0.1 mM, were similar to those of GLUT5−/ − mice, and did not cause hyperglycemia. Fructose levels more than doubled in WT mice fed isocaloric 20% fructose, causing significant hyperglycemia. Fructosemia and hyperglycemia in rats also increased dose‐dependently with dietary fructose. Deletion of mouse FK prevented fructose‐induced hyperglycemia, but caused dramatic hyperfructosemia (>1 mM) that not only worsened with fructose dose but also surprisingly reversed portal to systemic fructose concentration gradients. Systemic glucose increased by 3.5 mM for each 0.1 mM increase in systemic fructose levels in WT, but was independent of systemic fructose in FK−/−, mice. It did not vary with portal fructose in WT and FK−/− mice, suggesting an important role of the liver in fructose‐induced hyperglycemia. Chronic fructose feeding of > 30 d also increased hyperglycemia and HbA1c concentrations in rats and WT mice, but not in FK−/− mice. Even after 12 wk of fructose feeding, hyperfructosemia was maintained in FK−/− mice which appeared phenotypically normal. Significant levels of blood fructose, perhaps from endogenous synthesis, were observed in rats and in WT, FK−/− and GLUT5−/− mice that were not fed fructose. FK−/− mice mimics the hyperfructosemia of essential fructosuria in humans. In WT mice, FK prevents the modest hyperfructosemia that seems to cause fructose‐induced hyperglycemia and diabetes in mammals.

Support or Funding Information

NSF 1121049, 1456673; NIH‐DK‐102934
Effects of Frying in Various Cooking Oils on Fatty Acid Content of Farmed Rainbow Trout (Oncorhynchus Mykiss)
Authors: Flaskerude Katrina , Picklo Matthew , Johnson LuAnn , Raatz Susan
Published: June 2021

Our goal was to describe the effects of frying with various oils on the fatty acid content of rainbow trout. Four different oils were evaluated (peanut oil, high oleic sunflower oil, corn oil, and canola oil). Farmed rainbow trout (Oncorhynchus mykiss) fillets were sliced into three portions and each section was trimmed to a 50 g piece for cooking and a 5g raw portion. Fillets were individually cooked with each of the oils (peanut oil, high oleic sunflower oil, corn oil, and canola oil) in an electric frying pan until the core temperature reached 63°C (145°F). The sample was set aside until cool to the touch and then re‐weighed. After frying, both raw and fried samples were frozen at −80°C until processing for analysis. Frozen samples were pulverized, and lipids were extracted from the samples using a Folch extraction. The total fatty acids of the samples were determined by gas chromatography after the fatty acids were converted to fatty acid methyl esters. Frying in high oleic sunflower oil provided the greatest difference in fatty acid composition with a significantly decreased 14:0, 16:0, 18:0, 18:2 n‐6, 18:3n‐3, 20:5n‐3, 22:5n‐3, and 22:6n‐3. 14:0 and 18:3n‐3 were significantly lower in fish fried in peanut oil, when compared to raw fish. There was no significant difference between the fatty acid concentrations raw fish compared to fish fried in corn oil or canola oil. Peanut oil reduced the 14:0 and 18:3n‐3 content of the fish. Frying in high oleic sunflower oil caused the greatest changes in fatty acid content of the fillets.

Support or Funding Information

USDA, NIFA 2014‐67017‐21758 and USDA 3062‐51000‐053‐00D.