bluuebunny/crossref_metadata_2025 huggingface dataset:
Quercetin aglycone up‐regulates antioxidant response genes and proteins in osteoblast‐like fetal rat calvarial cells
Authors: Messer Jonathan G. , Hopkins Robin G. , Kipp Deborah E.
Published: June 2021

Quercetin (QRC), a flavonol found in fruits and vegetables, has been shown to up‐regulate antioxidant response genes and proteins in a variety of cell types, but little is known about the effects of QRC on these genes and proteins in osteoblasts. The purpose of this study was to determine the extent that QRC alters expression of antioxidant genes and proteins in primary cultures of fetal rat calvarial cells, a well‐described model of osteoblast differentiation. Cells were treated up to 48 h starting at confluence with 0 or 20 μM QRC aglycone. Antioxidant gene and protein expression were analyzed using real‐time quantitative PCR and Western blotting, respectively. Quercetin significantly up‐regulated heme oxygenase 1 (HO‐1), catalase, and gamma‐glutamate‐cysteine ligase catalytic subunit (GCLC) (p < 0.05), but did not up‐regulate peroxiredoxins 3 and 5, and NADPH:quinone oxidoreductase (p >; 0.05). Protein expression was generally congruent with gene expression, with HO‐1 being the most markedly up‐regulated in response to 20 μM QRC. These data suggest that QRC up‐regulates some genes and proteins associated with the antioxidant response in osteoblasts. Since evidence suggests that oxidative stress may contribute to osteoporosis, QRC may have protective effects on bone health.

Grant Funding Source: USDA Agricultural Research Services
Intermittent Bolus Feeding Enhances Lean Tissue Accretion by Increasing Muscle Amino Acid Transport and Protein Turnover Compared with Continuous Feeding in Neonates
Authors: El‐Kadi Samer , Boutry Claire , Suryawan Agus , Gazzaneo Maria C. , Orellana Renan A. , Srivastava Neeraj , Nguyen Hanh V. , Fiorotto Marta L. , Davis Teresa A.
Published: June 2021

Orogastric tube feeding is recommended in neonates unable to ingest food normally, and can be administered by intermittent bolus (INT) or continuous (CON) infusion. Recently, we showed that INT compared to CON feeding enhances lean tissue accretion by increasing muscle protein synthesis in neonates. To determine the mechanism for this response, neonatal pigs (n=6, 6‐d‐old) were fed the same diet in equivalent amounts continuously or intermittently (meal every 4h) for 21d. Insulin receptor and insulin receptor substrate 1 expression and protein kinase B phosphorylation in skeletal muscle were unaffected by feeding modality. Formation of the active eukaryotic initiation factor (eIF) 4E‐eIF4G complex and phosphorylation of ribosomal protein S6 kinase were higher and phosphorylation of eIF2α was lower in INT compared to CON fed pigs indicating increased translation initiation (P<0.05). Sodium‐coupled neutral amino acid transporter 2, but not L‐type amino acid transporter 1, expression was higher in INT compared to CON pigs suggesting enhanced glutamine transport (P<0.05). Abundance of the ubiquitin ligases, muscle RING finger‐1 and F‐box protein atrogin‐1/MAFbx, was higher for INT compared to CON pigs (P<0.05). These results suggest that intermittent feeding enhances lean tissue accretion by increasing amino acid transport and protein turnover. (Supported by NIH AR444474 and USDA/ARS 6250–51000‐055)
Lean Gain Is Enhanced by a Leucine Pulse during Long‐Term Continuous Feeding in Neonatal Pigs
Authors: Boutry Claire , El‐Kadi Samer W. , Suryawan Agus , Steinhoff‐Wagner Julia , Stoll Barbara , Orellana Renán A. , Nguyen Hanh V. , Davis Teresa A.
Published: June 2021

Infants who are unable to maintain oral feeding can be nourished continuously by orogastric tube. We have previously shown in neonatal pigs that a leucine pulse administered during continuous feeding enhanced protein synthesis in skeletal muscle compared to continuous feeding alone for a 24 h period. Twenty‐three piglets (7‐d‐old) were fed continuously a milk replacement formula via orogastric tube for 21 d with an additional parenteral infusion of either leucine (800 μmol•kg−1•h−1, LEU) or alanine (ALA) for 1 h every 4 h. At 21 d, the body weight was higher and the weight gain greater in LEU than in ALA pigs. The lean gain, determined by dual‐energy X‐ray absorptiometry (DEXA), was higher and the fat gain lower in the LEU than ALA group. Longissimus dorsi (LD), gastrocnemius and soleus muscle and kidney weights were heavier in LEU than ALA with no effect of treatment on liver, small intestine and heart weights. Fractional protein synthesis rate and formation of the active eukaryotic initiation factor (eIF) eIF4E•eIF4G complex in LD were greater in the LEU than ALA group. In conclusion, administration of a leucine pulse during 21 d of continuous feeding decreases fat gain and increases lean gain by stimulating the translation initiation pathway leading to protein synthesis in skeletal muscle in neonates. (NIH AR444474 and USDA/ARS 6250–51000‐055)

Grant Funding Source: NIH AR444474 and USDA/ARS 6250–51000‐055
Seed coat removal of red lentils enhances iron bioavailability in traditional Bangladeshi meals
Authors: DellaValle Diane M. , Vandenberg Albert , Glahn Raymond P.
Published: June 2021

Iron (Fe) deficiency is the most prevalent nutrient deficiency in the world, a major cause of which is a lack of highly bioavailable dietary Fe. Lentils (Lens culinaris) are a pulse crop consumed world‐wide, and are higher in bioavailable Fe compared to other legumes. We screened numerous meal combinations (n=46) for Fe concentration and relative Fe bioavailability using the in vitro digestion/Caco‐2 cell model. Traditional Bangladeshi dal was prepared with either dehulled (seed coat removed) or whole red lentil (seed coat intact). Other components of the meals included Basmati rice, vegetable curry, and steamed fish. Fe concentration of the mixed meals ranged from 4.8 – 65.0 ppm Fe. We observed a two‐fold increase in iron bioavailability in recipes containing dehulled lentil (p<0.001). Fe concentration and relative bioavailability were significantly correlated in the meals containing the dehulled lentil (r=0.52, p=0.01), but not in those containing whole lentil (r=−0.35, p=0.11). Since most red lentils are consumed with the seed coat removed, this class of lentils has potential to be a significant source of dietary Fe. These experiments highlight the need to improve our understanding of the effect of seed coat polyphenolics on Fe biovailability. Funded by the Saskatchewan Pulse Growers and USDA‐ARS.
Maternal anemia modifies the effect of multiple micronutrient supplements on birth weight and infant mortality in Indonesia
Authors: Sebayang Susy Katikana , Pierce Marcella , Harefa Benyamin , Aditiawarman Aditiawarman , Apriatni Mandri S. , Shankar Anita V. , Dibley Michael , Kelly Patrick J. , Tjiong Roy , Anwar Khaerul , Shankar Anuraj H.
Published: June 2021

Objective

To assess effects of maternal anemia on the impact of maternal multiple micronutrient (MMN) supplementation compared to iron & folic acid (IFA) in a double blind RCT in Indonesia (ISRCTN34151616).

Methods

We analyzed 14611 singleton births with data on maternal hemoglobin (Hb), height, parity, education, gestational age at birth, sex, season of birth & household wealth. Mortality was analyzed with Cox proportional hazards models; birth weight (BW) & maternal Hb with linear models; low birth weight (LBW), small for gestational age (SGA) & preterm‐LBW with logistic models.

Results

In anemic women MMN reduced early infant mortality (< 90 days of birth) by 39% [Adj HR 0.61; 95%CI 0.49–0.78 p<0.001; interaction (int) p=0.04] and neonatal mortality by 36% (Adj HR 0.64; CI=0.47–0.86 p=0.003; int p=0.18). MMN improved BW by 37g (CI −1 to 74 p=0.056; int p=0.01), and reduced LBW by 17% (aRR 0.83; CI 0.66–1.05 p=0.11; int p=0.04) and preterm‐LBW by 40% (aRR 0.60; CI 0.42–0.87 p=0.007; int p=0.002), and improved Hb by 0.28g/dl (CI −0.031 to 0.59 p=0.08) in anemic women.

Conclusions

Maternal MMN supplements, as compared to IFA, have greater benefits on infant survival and birth weight in anemic than in non‐anemic women.

Funding:

Turner Foundation, UNICEF, CHHD, USAID Indonesia grant # 497G00010000100. AusAID supported SKS's PhD at U of Sydney.
Relationship between psychosocial indicators and adoption of farm and garden activities and local produce in early childcare
Authors: Wickham Catherine , Cohen Nancy L. , Violette Catherine
Published: June 2021

Farm to Preschool Programs link farmers with early childcare centers (ECC) to increase local fruits and vegetables (LFV) used. To target nutrition and food safety education, it is important to identify factors related to adoption of farm and garden (FG) activities and LFV use in ECCs. An online survey was sent to 600 random ECC in MA and NH, with 118 educator (E) responses. Psychosocial factors related to LFV (benefits, barriers, self‐efficacy) were rated 1–5. FG activities and % of produce served as LFV were self‐reported, and related to psychosocial factors. Half of E did not know if FV were local and of those that did know, over half reported < 25% LFV. Most E perceived benefits of LFV and few barriers; however >;40% E perceived that LFV are too expensive. Overall self‐efficacy was low, with only 21% of E agreeing that they can get most of the LFV they want all year. About half of the sites had taken children on farm field trips and planted a garden; over 30% had used LFV in snacks or taste tests; few had invited a farmer to the ECC or held a harvest festival or farmers market. Perceived benefits and self‐efficacy were related to % of LFV served. Fewer barriers and higher benefits were related to more classroom FG activities. Nutrition education should thus address ways to increase perceived benefits, decrease barriers and improve self‐efficacy to LFV. Food safety education should address issues of FG visits, gardens, snacks and tasting LFV.

Grant Funding Source: This project is supported by The National Integrated Food Safety Initiative (NIFSI) Competitive Grant Program from the USDA National Institute of Food and Agriculture, Award 2011–51110‐30996.
Food insecurity in early life and BMI z‐score change among children from 2 to 15 years
Authors: Demment Margaret Mochon , Olson Christine M.
Published: June 2021

Food insecurity disproportionately affects children and families at highest risk for obesity. Initially perceived as separate health problems, there is growing concern that food insecurity and obesity are related. Longitudinal studies that examine the relationship food insecurity and weight status in children have found contradicting and inconclusive results. We hypothesized that the timing and transitions in food insecurity have different consequences for risk of developing obesity. Longitudinal data from a rural New York State birth cohort (n=517) were used to determine whether the rate of change in BMI z‐score in childhood, 2 to 15 years, differ by exposure food insecurity at birth and 2 years of age. Linear mixed models were used to estimate the rate of change in BMI z‐score during childhood. We found that food‐insecure children compared to food‐secure children, were smaller in early childhood. Due to a greater rate of change in BMI, by the time food‐insecure children reach 15 years of age they had higher BMI z‐scores. We provide evidence that children who experience food insecurity early in life have higher rates of change in BMI compared to food‐secure children, putting them at greater risk for overweight and obesity in adolescence. Our findings provide new insight into why longitudinal studies in early childhood have been inconclusive.

Grant Funding Source: Support for this project was provided by a Dissertation Award from the Robert Wood Johnson Foundation through its national program Active Living Research (ALR). Support for this student is provided in part by USDA National Needs Graduate Fellowship Competitive Grant No. 2008–38420‐04825 from the National Institute of Food and Agriculture. The data on biological and early life risk come from a NIH‐funded study No. HD 29549, “Biosocial Influences on Postpartum Weight Retention.”
Parenting Picky Eaters: Strategies to Get Preschool Children to Eat Previously Rejected Foods (PRF)
Authors: Antono Amanda C. , Goodell L. Suzanne , Daniel Joylyn C. , Walker Samantha L. , Johnson Susan L. , Power Thomas G. , Hughes Sheryl O.
Published: June 2021

Food neophobia affects a child's willingness to try new foods and can result in children consuming less diverse diets. Researchers conducted 17 focus groups (total participants = 103) to identify the strategies that parents use to get their preschool children to eat previously rejected foods (PRF). These focus groups targeted low‐income African American and Hispanic parents with young children (ages 3–5 years) in Texas, Colorado, and Washington. Using principles of grounded theory to analyze transcripts, researchers determined that parents most often intentionally decided not to purchase or serve PRF to their children. However, when parents did elect to serve PRF to their children, they employed strategies including serving the PRF in an alternative style (e.g. steaming instead of serving raw), adding an ingredient to the PRF (e.g. ranch dressing, cheese, sugar, or ketchup), involving children in the food preparation process, “tricking” the child to eat the PRF (e.g. telling the child that fish is chicken), and requiring the child to eat PRF. Results from this study will be used to create an intervention to help parents get their preschool children to eat PRF. Future investigations should focus on uncovering factors that motivate parents to repeatedly offer their children PRF, a key step in overcoming food neophobia.

Grant Funding Source: USDA Grant No. 2011–68001‐30009; USDA/ARS Cooperative Agreement No. 58–6250‐0–008
S‐adenosylmethionine Lowers Inflammatory Response in Human Monocytic Cells (THP‐1) and Alters DNA Methylation
Authors: Pfalzer Anna C , Choi Sang‐Woon , Park Lara K , Bottiglieri Teodoro , Lamon‐Fava Stefania
Published: June 2021

S‐adenosylmethionine (SAM), the unique methyl donor in DNA methylation, has been shown to lower inflammation. To assess whether epigenetic mechanisms mediate this effect, THP‐1 cells were differentiated into macrophages and treated with 0 μmol/L, 500 μmol/L(low), or 1000 μmol/L (high) SAM for 24‐hours, followed by stimulation with LPS. TNFα and IL‐10 expression levels were measured using RT‐PCR. Cellular SAM and S‐adenosylhomocysteine (SAH), a metabolite of SAM, were measured by LC/MS/MS. DNA methylation was measured using LC/MS/MS and microarrays.

Relative to 0 μmol/L SAM, treatment with low SAM caused a significant decrease in TNFα expression (−45%, p<0.05) and increase in IL‐10 expression (+77%, p<0.05). Treatment with high SAM yielded no significant additional benefits. Relative to 0 μmol/L SAM, low SAM treatment increased cellular SAM concentrations 2‐fold (56 nmol/mg vs 90 nmol/mg) without changes in SAH. Treatment with high SAM increased cellular SAM 6‐fold (330 nmol/mg) and SAH 4‐fold (2.1 nmol/mg vs 9.2 nmol/mg) relative to control. Genomic DNA methylation was significantly increased from 4.5% to 4.8% (p<0.05) only by treatment with low SAM. Microarrays identified 765 differentially methylated regions.

SAM exerts significant anti‐inflammatory effects through modulation of gene expression, possibly mediated by DNA methylation.

Funded by USDA ARS contract#1950–51000‐072 and by Pharmavite LLC

Grant Funding Source: USDA ARS contract #1950–51000‐072 and Pharmavite LLC
Dietary sulforaphane elicits off‐target effects at loci coding for long terminal repeats in lymphocytes from healthy adults and in IMR‐90 fibroblast cultures, possibly impairing genome stability
Authors: Baier Scott R , Zbasnik Richard , Schlegel Vicki , Zempleni Janos
Published: June 2021

Previous studies suggest that sulforaphane (SFN), an isothiocyanate in cruciferous vegetables, inhibits histone deacetylases (HDACs), thereby activating the tumor suppressor genes p21 and Bax and decreasing cancer risk. We posit that HDAC‐dependent effects of SFN are not specific for tumor suppressor genes. We tested the hypothesis that SFN elicits undesirable off‐target effects through the activation of repressed loci such as long terminal repeats (LTRs). De‐repression of LTRs impairs genome stability. We tested our hypothesis in broccoli sprout feeding studies in adults using a randomized crossover design and in SFN supplementation studies in human IMR‐90 fibroblast cultures. The mean abundance of LTR mRNA was 36% greater in SFN‐supplemented (2.0 μM SFN) fibroblast cultures compared with low dose SFN (0.5 μM) and SFN‐free controls (as per qRT‐PCR; P=0.03; one‐way ANOVA). The same dose‐response pattern was seen for the mean LTR mRNA abundance in lymphocytes in human feeding studies: 472% (102 g sprouts), 157% (68 g sprouts), and 100% (no sprouts)(0.06<P<0.19). We conclude that effects of SFN on gene activity are not specific for tumor suppressor genes and propose that caution needs to be exercised when recommending supplementation with high doses of SFN. It is unknown whether off‐target effects of SFN outweigh the beneficial effects of SFN.

Grant Funding Source: ARD Hatch, NIFA, and NIH
Epigenetic synergies between methyl donors and biotin in gene repression are mediated by holocarboxylase synthetase (HLCS)
Authors: Xue Jing , Wijeratne Subhashinee S.K. , Zempleni Janos
Published: June 2021

HLCS catalyzes the binding of biotin to proteins in humans. Previously, we have shown (1) that HLCS interacts physically with the methyl transferase EHMT‐1, which creates histone H3 K9 methylation (H3K9me) gene repression marks, (2) that biotinylation of EHMT‐1 strengthens interactions with HLCS, (3) that biotin depletion and HLCS knockdown cause a loss of H3K9me marks leading to de‐repression of long terminal repeats (LTRs) and to chromosomal abnormalities, and (4) that erasure of DNA methylation impairs HLCS‐dependent biotinylation events. Here, we tested the novel hypothesis that methyl donors and biotin synergize in mediating gene repression through epigenetic marks, and that HLCS contributes toward mediating these synergies. Co‐immunoprecipitation (Co‐IP) and limited proteolysis assays (LPA) confirmed that HLCS interacts physically with the DNA methyl transferase DNMT1 and methyl CpG binding protein MeCP2. Overexpression of HLCS in HEK293 cells caused a 200% increase of H3K9me marks in LTRs (Kruskal‐Wallis, P<0.001), judged by ChIP, and an 84% decrease in LTR mRNA (t‐test, P<0.05), judged by qRT‐PCR, compared with controls; erasure of DNA methylation by azacytidine abrogated effects of HLCS. We conclude that interactions between HLCS and chromatin proteins create epigenetic synergies between biotin and methyl donors in gene repression, thereby maintaining genome stability.

Grant Funding Source: ARD Hatch, NIFA, NIH
Storage stability of provitamin A carotenoids in Biofortified yellow maize
Authors: Muzhingi Tawanda , Tang Guangwen
Published: June 2021

Biofortification increases the micronutrient content of staple crops through plant breeding. Maize is a staple crop in Africa and Latin America where vitamin A deficiency (VAD) is also a public health problem. Provitamin A carotenoids such as α‐carotene (α‐C), β‐cryptoxanthin (β‐Cr) and β‐carotene (β‐C) in Biofortified Yellow Maize (BYM) may lead to a reduction in the prevalence of VAD. However, carotenoids degrade when exposed to excessive light, moisture, and heat. It is imperative to establish the extent of post‐harvest losses of carotenoids in BYM under simulated tropical storage conditions. BYM maize was stored for 4 months at I) ‐ 80°C in the freezer, II) 4°C in the fridge, III) 25°C + 50% humidity in an incubator and IV) 20°C at room conditions. The provitamin A carotenoid contents were determined at baseline and monthly for 4 months by HPLC with a C30 column. BYM β‐C content at baseline was 15.2 ±0.6 μg/g dry wgt. At endline the β‐C content loss was 18.6, 52.7, 73.9 and 79.5% for conditions I–IV. The α‐C content loss was 23.4, 35.0, 57.0 and 63.9% for conditions I–IV. For β‐Cr, the loss was 10.6, 30.1, 46.1 and 57.6 % for conditions IIV, respectively. The provitamin A carotenoid losses in BYM are >; 45% at temperatures >; 20°C. These losses should be addressed to retain the vitamin A value of BYM over long periods of storage under tropical conditions, in order to control VAD effectively.

Grant Funding Source: USDA contract # 58–1950‐0–014
Using the novel RADR mouse to visualize the effects of age and environment on DNA repair in vivo in multiple tissues
Authors: Jackson Michelle R Sukup , Rowland Elizabeth A , Kay Jennifer E , Schulte Kelly , Chow Danielle N , Belanger Crystal L , Laura Trudel J , Matsuguchi Tetsuya , Wogan Gerald N. , Erdman Susan E , Engelward Bevin P
Published: June 2021

Homologous recombination (HR) is a DNA repair process that is critical for maintaining genomic integrity. HR is necessary for survival of vertebrate cells, but over‐ or under‐utilization of HR can lead to deleterious rearrangements and cancer. To study HR in vivo, we created the RADR (Rosa26 Direct Repeat) mouse that has enabled study of HR in a variety of tissues. The RADR mouse harbors two truncated EGFP genes integrated in the Rosa26 locus. Repair via HR at the substrate can yield a full‐length EGFP gene, resulting in a fluorescent cell. The frequency of HR can be estimated by flow cytometry or visualized in situ. The RADR mouse enables studies of DNA damage and repair in response to endogenous and exogenous factors in multiple tissues, which has never before been possible. We observed the accumulation of recombinant cells in the colon, liver and pancreas with age; this correlates with our understanding of age as a risk factor for cancer. We have also crossed the RADR mice with GPT‐Δ mice, enabling quantification of point mutations and small deletions. We crossed these mice with Rag2−/− mice allowing the study of sequence changes with enhanced innate immune response. In ongoing studies, we are investigating the impact of environmentally induced inflammation on susceptibility to large‐scale sequence rearrangements, point mutations and small deletions in multiple tissues. This work is supported by the NCI, NIEHS, NSF GRF.

Grant Funding Source: NSF GRF, NCI, NIEHS
Mechanical properties of intramandibular sutures in the American alligator
Authors: Zapata Uriel , Elsey Ruth M. , Wang Qian
Published: June 2021

The skull is a complementary combination of flat bones joined by connective suture tissues. Although brain growth produces mainly quasi‐static tension loads across the sutures, physiological loads could produce an alternate combination of tension‐compression, thus tension test looks to be the best way of estimate differences in the biomechanical behavior of sutures. We hypothesized that, [1] the mechanical properties of sutural tissue in the America alligator (Alligator mississippiensis) changed during ontogeny, and [2] there were sex‐based difference in the change during ontogeny. Modulus of elasticity (E), and maximum normal elastic stress (σy) were evaluated by using a tension test technique in three different mandibular sutures and a control group of mandibular cortical tissue. 67 specimens were removed from 15 subject (8 males and 7 females) covering three ontogenetic groups: [1] juvenile from 12–36 in. long, [2] junior adult from 37–72 in. long, and [3] senior adult from 73–96 in. long. Results demonstrated that there were no significant differences in mechanical properties between male (E=12.87 ± 15.79 MPa) and female animals (E=12.41 ± 13.55 MPa). However, there were significant differences in elastic modulus between specimens from juvenile group and specimens from adult age groups (p<0.05). In addition, there are statistical differences not only between the control group and the specimens including sutures, but also among sutural positions. In conclusion, mechanical properties are associated with physiological conditions that could affect sutural function, and some mechanical properties in mandibular sutures decrease during ontogeny, suggesting a direct effect of mineralization processes in mechanical properties.

Grant Funding Source: Supported by NSF BCS‐0725183, and COLCIENCIAS (Bogotá, Colombia)
Lemur biorhythms and life history evolution
Authors: Hogg Russell , Godfrey Laurie , Schwartz Gary , Dirks Wendy , Bromage Timothy
Published: June 2021

Microstructural data from the skeleton support the hypothesis that primate life histories are centrally regulated by a neuroendocrine rhythm, the Havers‐Halberg Oscillation (HHO). However, subfossil lemurs are outliers in HHO scaling relationships which have been discovered for primates and mammals in general. We present new data to determine whether these species represent the general strepsirrhine condition and to inform general models about neuroendocrine‐mediated life history evolution. We gathered the largest sample of HHO data from histological sections of primate teeth to date, regressing them against life history related variables such as body mass and basal metabolic rate. For all primates, mass and metabolic variables show strong and significant correlations with HHO in line with predictions. However, strepsirrhines do not follow this pattern when examined separately. Therefore, phylogeny may play an unforeseen role in governing how HHOs of specific taxa respond to ecological forces; in the case of the lemurs, which have been subjected to isolated ecologies due to living in Madagascar, these effects seem to have been especially potent. This reinforces the idea that HHO influences life history evolution in response to specific ecological selection regimes. This project was funded by NSF grants BCS‐0622479 (RTH), BCS‐0237338 and BCS‐0503988 (LRG and GTS), and 2010 Max Planck Research Award (TGB).
Internal Bone Structure and Orientation in the Browridge of Homo, Pan, Gorilla, Papio, and Cebus
Authors: Smith Leslie Claire Pryor , Dechow Paul C.
Published: June 2021

Assessing the variation of the internal bone structure, anisotropy and orientation in the browridge across regions and species can aid in our understanding of craniofacial adaptation. Also, quantifying species and region specific variation may serve to improve the accuracy of finite element models. In this study, μCT was used to determine the structural properties, degree of anisotropy (DA) and primary trabecular orientation in 3 equally divided browridge regions of 8 Homo, 5 Pan, 7 Cebus, 4 Papio, and 1 Gorilla. Both bone volume fraction (BVF) and degree of anisotropy (DA) differed significantly by species (Kruskal‐Wallis Test, p<0.001 and p<0.015, respectively). Most regions across species showed orthotropy. In all species and regions, trabeculae tend to show less variability in orientation across transverse and coronal planes. Orientations in the sagittal plane instead show higher variability across individuals. The results suggest that BVF, DA and orientation of trabecular‐like bone within the supraorbital region may be reflective of feeding strategies and that characterization of subcortical structure in craniofacial regions may improve finite element models. Additional study of trabecular bone orientation within zygomatic regions is ongoing to examine how trabecular bone orientation and DA differ across the entire craniofacial region in these species.

Grant Funding Source: Science Foundation Physical Anthropology HOMINID program (NSF BCS 0725141)
Novel anatomical analysis of human embryos using iodine staining and micro‐computed tomography (CT Scanning)
Authors: Spaw Alexandra , Witmer Lawrence M.
Published: June 2021

Human developmental anatomy is central to understanding of many disease states but has been challenged by the typically destructive analysis (e.g., histology) of scarce fetal specimens. CT scanning is non‐destructive, but is usually poor at distinguishing soft tissues. Iodine potassium iodide (I2KI) staining prior to CT is a new method to enhance soft‐tissue contrast. This study is the first to study human development with iodine‐enhanced microCT. Six human embryos ranging from 6–18 weeks, will be scanned, soaked in Lugol's solution for days to months depending on size, and then rescanned. Data are analyzed in Avizo and presented as images and 3D models. Here we present a progress report based on a subset of the total sample. Iodine staining shows many distinguishable soft tissues and provides highly detailed images. Results include clear details of the developing brain, fetal heart and circulation, and gut. The contrast provided by iodine staining creates images that can be used to analyze components of fetal anatomy that can provide insight into many developmental anomalies. This initial success indicates that this technique has great promise for producing valuable visualizations of human fetal development and a better understanding of human disease. Funding for this project was provided by a grant to AS from the Ohio University Provost Undergraduate Research Fund and to LMW from NSF.

Grant Funding Source: NSF
UmuD participates in a primitive DNA damage checkpoint by interacting with DNA pol III α and SSB
Authors: Silva Michelle Cristine , Chaurasiya Kathy R , Ruslie Clarissa , Voortman Lukas , Ronayne Erin A , Nevin Philip , Lone Samer , Chan Monyrath , Williams Mark C , Beuning Penny J
Published: June 2021

The α subunit of DNA polymerase III (DNA pol III) is the main replicative polymerase in Escherichia coli. Because α cannot copy damaged DNA, replication is stalled in presence of DNA lesions. To compensate, the cell initiates the SOS response, inducing the expression of at least 57 genes. The products of these genes are involved in DNA damage tolerance mechanisms, one of which is translesion synthesis (TLS), a process by which lesions are bypassed by specialized DNA polymerases with the ability to copy damaged DNA. Full‐length UmuD, whose expression is regulated by the SOS response, inhibits DNA replication and prevents mutagenic TLS as part of a primitive DNA damage checkpoint, while the cleaved form UmuD’ facilitates mutagenesis. It has been suggested that UmuD participates in a primitive DNA damage checkpoint but its exact contribution is still unclear. In order to understand the role of UmuD in regulating DNA replication, the interactions between UmuD and α and between UmuD and SSB were investigated, the latter of which is a novel interaction. As a result, we have determined that UmuD disrupts the interaction between α and the β clamp, as well as the interaction between α and ssDNA, suggesting a direct role for UmuD in disrupting replication. The UmuD‐SSB interaction may contribute to the transition between the replication of undamaged DNA and TLS. Supported by NSF.
DNA polymerase phylogeny recapitulates virioplankton biology
Authors: Schmidt Helen Frances , Sakowski Eric G , Williamson Shannon J , Polson Shawn W , Wommack K Eric
Published: June 2021

Despite decades of investigation, we know little of the predominant biological characteristics of aquatic viruses that drive the rapid turnover of abundant virioplankton assemblages. DNA polymerases are essential to replication and thus enable connections between diversity and an important biological feature of viruses. Novel DNA polymerases, containing all critical functional domains, were common within dsDNA and ssDNA virioplankton metagenomic libraries. Finding DNA polymerases in ssDNA libraries was unexpected, as these genes have not been observed within ssDNA viruses. Comparison of PCR‐amplified environmental DNA pol sequences with virioplankton metagenome sequences showed that the majority of the metagenomic polymerase sequences grouped away from previously identified groups of viral DNA polymerases forming a new group that included siphoviruses. An F762L amino acid substitution was observed within this dominant clade. Phylogenetic analysis of phages with known life cycles indicates that an F/Y762L substitution is a strong predictor of lysogeny, indicating that slower‐replicating, lytic or lysogenic phage populations may predominate within the virioplankton and shape the influence of viruses on marine biogeochemical cycles. This work was funded by the HHMI Undergraduate Science Education program the NSF Microbial Genome Sequencing Program, the Gordon & Betty Moore Foundation, and Delaware EPSCoR.
Differential contacts of the 4.5 Myb repeats of SNAP190 with the DNA of U1 and U6 snRNA gene promoters
Authors: Stumph William E , Kang Yoon Soon , Kurano Michelle
Published: June 2021

U1 and U6 snRNA genes are transcribed by RNA polymerases II and III respectively, yet the transcription of each is dependent upon the small nuclear RNA activating protein complex (SNAPc). In D. melanogaster, DmSNAPc recognizes a 21 base‐pair DNA sequence, the PSEA, located approximately 40–60 base pairs upstream of the transcription start site of each gene. DmSNAPc is a heterotrimer of subunits named DmSNAP190, DmSNAP50, and DmSNAP43. All three subunits are required for DNA binding activity, but only DmSNAP190 contains a “canonical” DNA binding domain that consists of 4.5 tandem Myb repeats. Myb repeats are each ~50 amino acid residues in length and were first identified as forming the DNA binding domain of the Myb oncoprotein. By using a site‐specific protein‐DNA photo‐cross‐linking assay combined with site‐specific protein digestion, we have been able to map where each of the 4.5 Myb repeats of DmSNAP190 contact the DNA of the U1 and U6 PSEAs. Although many of the contact points are similar, the Myb repeats of DmSNAP190 interact with the U1 DNA primarily through the major groove, but new and distinct Myb repeats‐DNA contacts that span the minor groove are observed when DmSNAPc binds to a U6 PSEA. This conformational shift in DmSNAP190 binding is likely important in determining the RNA polymerase specificity of the U1 and U6 gene promoters. (Supported by NSF and in part by the California Metabolic Research Foundation.)
Systems Biology approach to identify therapeutic targets in nonhuman primates exposed to Y. pestis
Authors: Hoke Allison Violet , Gautam Aarti , Dimitrov George , Hammamieh Rasha , Jett Marti
Published: June 2021

Yesinia pestis, the etiological agent of the plague, induces vascular and pulmonary collapse in the host that is not well characterized. Y. pestis is known to have developed processes to overpower the host immune system and cause infections. In this study, a nonhuman primate model was exposed to aerosolized CO92 Y. pestis which has the potential to be used as a biological weapon. To understand the complex interaction between the pathogen and host, we have applied global ‐omics profiling to determine the basis of infections and identify host defense strategies and the mechanisms by which they are regulated. The genes, proteins, and metabolites profile show unique signatures shortly after exposure. In the infected monkeys, we identified a modified HIF‐1 pathway. We have shown profiles that have been generated from a few genes over the course of infection that are involved in molecular transport, immune response, and apoptosis. Quantitative Real‐Time PCR was utilized to validate the microarray results from blood chemistry profiles. The data from blood chemistry is also discussed. This research was supported through grant # TMTI0029_09_WR_T from the DoD Chemical and Biological Defense program through the Defense Threat Reduction Agency (DTRA).
Monofluorophosphate inhibits the key step of internal polysaccharide biosynthesis in Streptococcus mutans
Authors: Asención Diez Matias D. , Demonte Ana M. , Naleway Conrad , Iglesias Alberto A. , Ballicora Miguel A.
Published: June 2021

Streptococcus mutans is the leading cause of dental caries worldwide and accumulates a glycogen‐like internal polysaccharide (IPS) which contributes to cariogenicity when grows in excess of sugars. Previously, we characterized the ADP‐glucose (ADP‐Glc) pyrophosphorylase (ADP‐Glc PPase; EC 2.7.7.27) from S. mutans, belonging to the key step leading to IPS synthesis. The enzyme showed two active conformations, homotetrameric GlgC and heterotetrameric GlgC/GlgD, the latter being one magnitude order more active and having different regulatory properties. Also, sodium monofluorophosphate (MFP) is the toothpastes active compound with established anticariogenic properties, although the molecular target and/or its mechanism are still unknown. In this work, we show that MFP partially inhibits (up to a 70% inhibition) both ADP‐Glc PPase active conformations, with I0.5 values of 1.5 mM and 8 mM for GlgC/GlgD and GlgC, respectively. Interestingly, MFP completely inhibited (with an I0.5 3.2 mM) a Fru‐1,6‐bisP activated GlgC form. Controls with NaF up to 25 mM were conducted and no inhibitions were observed neither in GlgC nor GlgC/GlgD. The inhibition of ADP‐Glc PPase was correlated with in vivo studies showing a decreased IPS accumulation in S. mutans cultures in the presence of 2 mM MFP. Other in vivo implications of MFP concern decreases in lactate production, acidogenesis and bacterial growth. Considering that synthesis of glycogen occurs by different pathways in mammals and bacteria, it is worth to highlight that ADP‐Glc PPase can be visualized as a particular target for controlling S. mutans virulence by means of MFP. Supported by funds from Colgate‐Palmolive (CN and MAB), NSF MCB‐1024945 (MAB), and CONICET [PIP 2519], UNL [CAI‐Orientado and Redes] and ANPCyT [PICT’08 1754] (AAI)
Probing Ligand Induced Changes in Flexibility of Glutamate Dehydrogenase Using Collisional Quenching of Fluorescence
Authors: Li Chun , Bell Ellis
Published: June 2021

To probe the local environments of tryptophans in proteins we have developed a novel analysis of fluorescence collisional quenching using multiple emission wavelengths, combined with differently charged quenchers (acrylamide: neutral, Iodide: negative & Cesium: positive). With N Acetyl tryptophan, similar Stern‐Volmer constants are obtained at all wavelengths, as expected. With glutamate dehydrogenase, (4 tryptophans per subunit), 4 distinct families of constants are obtained with the highest occurring at long wavelengths and progressively decreasing as the wavelength band analyzed decreases showing this approach separates the effects on individual tryptophans in the protein. We have explored nucleotide effects on accessibility and charge environment of the 4 tryptophans. Results indicate that overall, nucleotide ligands NADH, NADPH and ADP significantly lower accessibility particularly in the region of subunit interfaces while GTP has less effect and in the case of W72 and W281 increases accessibility. Acrylamide quenching gives Stern‐Volmer plots concave upwards, which become linear when lifetime measurements are used. With NAD(P)H, the charged quenchers, Iodide and Cesium (but not the neutral Acrylamide) give negative slopes which appear to result from differential effects on tryptophan‐NAD(P)H resonance energy transfer. Funded by NSF Grant MCB 0448905 to EB
Determining the role(s) of prime‐side residues in macromolecular inhibition of trypsin‐fold serine proteases
Authors: St. Germain Commodore , Baird Teaster
Published: June 2021

Trypsin‐fold serine proteases are among the most abundant of all proteases. By identifying residues that are important for enzyme‐inhibitor interaction in trypsin, we may advance the development of protease‐based therapies in general. Lysine‐60 (K60) is a highly conserved prime‐side residue that may play many roles in trypsin. In co‐crystals, K60 hydrogen bonds to tyrosine‐39 (Y39) and is positioned to restrict conformational mobility of phenylalanine‐41 (F41), possibly limiting the observed hydrogen bond interactions between those residues and macromolecular inhibitors. Substitution of K60 with other amino acid residues may disrupt these interactions and provide insight to their significance with respect to inhibitor binding. To test this hypothesis, we created trypsin variants K60G, K60A, K60V, K60I, and K60R and characterized them with respect to their activities and sensitivities to the macromolecular inhibitors soybean trypsin inhibitor (SBTI) and bovine pancreatic trypsin inhibitor (BPTI). Our initial results show that, compared to wild‐type trypsin, K60A and K60V are catalytically indistinguishable, more resistant to autolysis and more sensitive to inhibition by SBTI and BPTI. Further experiments with ecotin and other trypsin variants can provide additional insight on specific interactions. This work was supported by NSF CAREER Award MCB‐0643988–02 and NIH MARC T34‐GM008574.
Imaging promoter activity to monitor gene expression using Intracellular Multiaptamer Genetic tag (IMAGEtag)
Authors: Ray Judhajeet , Shin Ilchung , Zhao Ming , Ilgu Muslum , Gupta Vinayak , Beasley Jonathan , Agrawal Aditi , Peng Leilei , Kraus George , Nilsen‐Hamilton Marit
Published: June 2021

Changes in gene expression play a central role in determining the physiological activities of cells. To understand the dynamics of gene expression several techniques have been developed recently but their applicability have been limited by factors like choice of reporter and target selection. We have developed an aptamer‐based imaging system to detect changes in promoter activity in living cells and in real time. In this technology we have cloned and expressed multiple copies of aptamers in tandem (viz. tobramycin or neomycin‐B or PDC) under inducible or constitutive promoters in Saccharomyces cerevisiae. The yeast cells were incubated with the respective ligands, which were conjugated to a Cy3 or a Cy5 dye. The promoter activities were then monitored by FRET (Förster resonance energy transfer), which occurred due to binding of the multiaptamers to the fluorophore labeled ligands. This technology allows the visualization of real time promoter activity from an inducible promoter like GAL1 by FLIM‐FRET, which was detected by the decrease of donor lifetime. We have been able to measure promoter activity from individual cells and found intercellular variation of gene expression upon induction. The IMAGEtag technology has the potential for application in measuring gene expression during various cellular events and for understanding it's distribution and temporal change in individual cells of the same type within multicellular or homogeneous populations.

Funded by National Institute of Biomedical Imaging and Bioengineering, U.S. Department of Energy, Office of Biological and Environmental Research
S. cerevisiae coq5 null mutants require over‐expression of Coq8 kinase for rescue by E. coli COQ5 homolog ubiE
Authors: Nguyen Theresa P.T. , Clarke Catherine F.
Published: June 2021

Coenzyme Q (Ubiquinone or Q) is an essential lipid electron carrier in the respiratory electron chain. In Saccharomyces cerevisiae, eleven genes (COQ1–9, ARH1, YAH1) are required for coenzyme Q biosynthesis, several of which encode proteins involved in a multi‐subunit complex. One of these genes, COQ5, encodes a C‐methyltransferase responsible for making the late stage intermediate demethoxy Q6 (DMQ6). COQ5 has three conserved methyltransferase motifs, and yeast point mutants within or adjacent to these motifs (coq5–2, coq5–5) lack Coq5 activity but retain steady state levels of the Coq5 polypeptide. We show that ubiE, the E. coli homolog for COQ5, is able to rescue coq5–2 and coq5–5 point mutants, but is unable able to rescue the yeast coq5Δ null mutant. However when putative kinase Coq8 is over‐expressed, ubiE is able to rescue the yeast COQ5 knockout. We demonstrate that co‐expression of COQ8 and ubiE in a coq5Δ null mutant restores Q6 levels and supports growth on non‐fermentable carbon sources. We also show the effects of complementation on the Coq multi‐subunit complex in the coq5 mutants via 2D Blue native‐PAGE. By observing the accumulation of Q intermediates in coq5 mutants, we have been able to elucidate steps within a novel Q biosynthetic pathway.

Studies supported by National Science Foundation Grant 0919609.
Immunization against hyaluronidase Hyal‐2 provides long‐term cancer prevention
Authors: Chang Nan‐Shan , Su Wan‐Pei
Published: June 2021

When immune deficient or competent mice were pre‐injected with micromolar levels of Zfra (a 31‐amino‐acid zinc finger‐like protein), these mice acquired a long‐term resistance to the growth and metastasis of a broad spectrum of cancer xenografts. Injected Zfra is accumulated mainly in the spleen, and, accordingly, the induced anticancer response is memorized and can be transferred to naïve immune‐deficient mice via spleen cells. Alteration of the Ser8 phosphorylation site to Gly8 abolished Zfra polymerization and cancer prevention in vivo. Zfra suppresses the activation of tumor suppressor WWOX (FOR or WOX1) and the expression of hyaluronidase Hyal‐2 in the spleen. Likewise, antibody against Hyal‐2 or activated WWOX mimics and dramatically boosts the anticancer effect of Zfra. Mechanistically, Zfra binds and activates non‐T/non‐B memory Hyal‐2+ spleen cells, via Hyal‐2/WWOX/Smad4 signaling, for conferring a long‐term protection against cancer growth and metastasis in vivo. (Supported in part by NHRI and DOD, Taiwan, and DoD, USA)
Physically modified hyaluronan in cancer prevention
Authors: Chang Nan‐Shan , Su Wan‐Pei
Published: June 2021

Although oligomeric HA may exhibit cancer‐blocking function, the functional role of hyaluronan (HA) in blocking or promoting cancer growth is largely controversial. When immune deficient or competent mice were pre‐injected with high molecular weight or native HA (medical grade), these mice became highly susceptible to the growth of melanoma and many cancer xenografts. However, when native HA was subjected to partial degradation and conformational alteration by appropriate physical approaches (e.g. UV irradiation, heat treatment, etc), one of the HA preparations, designated HAson, provided a long‐term prevention of the growth of skin cancer, melanoma, breast adenocarcinoma, and many other cancers in mice. A specific hyaluronidase Hyal‐2 antibody reproduced the induced cancer prevention. Further, HAson‐induced anticancer activity is memorized in the non‐T/non‐B Hyal‐2+ spleen cells, as the induced anticancer activity can be transferred to naïve immune‐deficient mice via spleen cells. Together, HAson binds and activates memory Hyal‐2+ spleen cells via the Hyal‐2/WWOX/Smad4 signaling to potently elicit long‐term cancer prevention in vivo. (Supported in part by NHRI and DOD, Taiwan, and DoD, USA)
Systematic deletion analysis of components in a glucose sensing pathway and the mating pathway in Saccharomyces cerevisiae points to significant interdependence
Authors: Klouda Elisabeth , Denny Joshua E , Duck Zachary A , Haskins David , Johnson Janelle L , Selcer Katie , Brigati Jennifer R , Willhite D. Grant , Wright Stephen E
Published: June 2021

Mating in Saccharomyces cerevisiae is an energy intensive process, so it follows that yeast cells would want to assess sugar availability prior to mating. Our lab has determined that optimal mating efficiency is dependent on a glucose sensing pathway mediated by the G‐protein coupled receptor, Gpr1. In this study we systematically assessed the impact of deficiencies in all known participants in the Gpr1 pathway on the mating pathway. The results indicated that many components of the glucose sensing pathway are necessary for optimal function of the mating pathway. To determine potential reciprocal effects, we assessed the impact of deficiencies in all known participants in the mating pathway on the glucose sensing pathway. The results indicated that the glucose sensing pathway was also dependent on many of the components of the mating pathway for complete activity. This analysis indicates significant interdependence between yeast mating and glucose sensing through Gpr1, and points to at least two levels of interaction between these two pathways. This research was funded by the National Science Foundation, Award #: 0952519.