bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of the Excluded Strand in DNA Unwinding by Hexameric Helicases
Authors: Trakselis Michael 
Published: June 2021

Hexameric DNA helicases control both the initiation and elongation phases of DNA replication. It is well known that these toroidal enzymes encircle and translocate preferentially on one strand with specific polarities dependent on their families and organismal domains. The question remains as to what if any role the excluded strand has in the unwinding mechanism or coordination of activities at the replication fork. Over the past few years, there has been an increasing amount of evidence to suggest that the excluded‐strand plays a significant role during unwinding by interacting with the helicase's outer surface in a Steric Exclusion and Wrapping (SEW) mode. The implications are that the excluded strand interaction controls the kinetics of unwinding while maintaining the ssDNA product for handoff to other replication components.We have investigated the interaction of the excluded strand with the outer surface of helicases in archaea, mitochondria, and bacteria using a variety of biochemical, biophysical, and genetic techniques. We have not only defined a minimal interaction path on the outer surface of these helicases, but we can also show that this interaction differentially controls the unwinding rate and efficiency. For the archaea MCM helicase, which has 3′‐5′ polarity, disruption of the excluded strand interaction inhibits unwinding consistent with a ratchet mechanism. Conversely, for the bacterial DnaB helicase, which has 5′‐3′ polarity, disruption increases the unwinding rate more consistent with a regulator‐type mechanism. The contrasting effects of the excluded strand interaction coupled with the opposing translocation polarities of these helicases will be discussed within the context of their respective replisomes.Support or Funding InformationNSF‐1613534
Cracking the Interorganellar Communication Codes
Authors: de Souza Amancio J , Svozil Julia , Wang Jin‐Zheng , Ke Haiyan , Xiao Yanmei , Gruissem Wilhelm , Dehesh Katayoon 
Published: June 2021

Inter‐organelle communication is essential for cellular homeostasis and stress adaptation. In plants, the metabolic hub is the chloroplast, which has a central role in tightly adjusting metabolic activities to environmental perturbations. Several small chloroplast molecules have been identified to be involved in signal transduction pathways that can modulate the transcription of nuclear genes. Despite its biological significance the underlying molecular mechanisms of chloroplast to nucleus signaling are still poorly understood. An intermediate of the plastid isoprenoid biosynthetic pathway (MEP), 2‐C‐methyl‐D‐erythritol‐2,4‐cyclopyrophosphate (MEcPP), has been shown to function as an inter‐organelle signal. Exogenous application of MEcPP or accumulation of this metabolite in Arabidopsis mutant plants (ceh1) results in induction of specific nuclear stress‐responsive genes. It has also been demonstrated that MEcPP accumulation potentiates the endoplasmic reticulum unfolded protein response, which is a critical component of stress adaptive responses. MEcPP accumulates in plants during environmental stress and/or physiological processes including high‐light, day‐night transitions, wounding, exposure to herbicides, and high‐temperature. It is well established that signaling events in plants involve the ubiquitin proteasome system (UPS). Plant hormones such as jasmonic acid, auxin and abscisic acid all rely on the UPS pathway for signal transduction. We have found that accumulation of MEcPP results in increased levels of ubiquitylation, raising the possibility of cross‐talk between the UPS and MEcPP in signal transduction processes. To address this possibility, we constructed Arabidopsis plants that accumulate high levels of MEcPP after induction and used a proteomics approach to detect MEcPP‐dependent ubiquitylation targets. This has identified 169 candidate proteins with increased ubiquitylation upon MEcPP induction. The results suggest that MEcPP signaling could be involved in reducing the activity of central metabolism and UPS‐mediated protein turnover. Additional experiments are being conducted to identify the ubiquitin E3 ligases activated by MEcPP signaling, which could be important targets for crop improvement and biotechnology.Support or Funding InformationNIH, NSF
Transition State Mimic of Intrinsic Hydrolysis in Ras GTPase
Authors: Ortiz Alicia Yvette 
Published: June 2021

Ras GTPases are involved in signal transduction pathways that control fundamental cellular processes such as proliferation, cycling, survival, and apoptosis. Ras functions via an on/off switch mechanism, where Ras is active in downstream signaling when bound to GTP and inactive upon GTP hydrolysis to GDP. Our lab discovered an allosteric site that is proposed to interact with the membrane, where binding in this allosteric site promotes ordering of the switch II region, favoring a conformation conducive to hydrolysis of GTP to GDP. When Ca2+ and acetate bind at this site in Ras crystals there is a disorder to order transition in switch II, positioning catalytic residues in place for hydrolysis. Interestingly, mutations of R97 to bulky aliphatic residues, such as L and M, mimic this effect, increasing hydrolysis rate constants relative to wild type. We had previously shown that in the presence of AlF3 RasR97L‐GDP binds to the Ras binding domain of Raf (Raf‐RBD), mimicking the γ‐phosphate of GTP. Here we present gel filtration studies of the R97M‐GDP mutant in the presence of Raf and AlF3 showing complex formation. The fact that Raf‐RBD binds to the R97L and R97M mutants, but not to wild type Ras, suggests that in these mutants the conformation associated with hydrolysis is more populated than in the wild type. AlFx has been used extensively to visualize the transition state of the GAP‐catalyzed GTP hydrolysis on Ras and its homologues. We are currently working on obtaining crystals of the H‐RasR97M‐Raf‐AlFx complex as a transition state model to elucidate the mechanism of intrinsic hydrolysis in Ras.Support or Funding InformationNSF MCB‐1517295
Application of a High‐Performance Liquid Chromatography Method for the Evaluation of Non‐Provitamin A Carotenoids
Authors: Titcomb Tyler James , Davis Christopher R. , Tanumihardjo Sherry A. 
Published: June 2021

Biofortification of maize with provitamin A carotenoids is an emerging intervention for providing vitamin A to people where maize is a staple crop. Analyzing the effectiveness of biofortified maize adoption will be important in the coming years. We propose that serum carotenoid profiles will be ideal for this endeavor. The C30 HPLC column was developed specifically for carotenoid separations because the longer alkyl chains provide better separation of carotenoids and their respected isomers. Most types of maize that have been released also have enhanced concentrations of the xanthophyll carotenoids lutein and zeaxanthin. Due to the chemical similarity of lutein and zeaxanthin, the C30 column is ideal for analysis because it is capable of maintaining separation of these two non‐provitamin A carotenoids. A potential drawback of the C30 column is longer analytical time specifically for lycopene. When monitoring study subject low carotenoid diets during a human maize feeding trial, we propose that lycopene can be used as a qualitative measure of compliance due to the abundance of the carotenoid in the typical western diet and the absence of lycopene in maize. Here we show the adaptation of an established HPLC method (Howe & Tanumihardjo, 2006) to include the quantification of lycopene and its respective isomers with a run time of 52 minutes, including equilibration, while maintaining the separation of lutein and zeaxanthin. Thus, serum carotenoids analysis could be useful for both measuring the adoption of maize and compliance in controlled feeding studies.Support or Funding InformationSupported by USDA Hatch WIS01804, Global Health Funds at UW‐Madison, and Harvest Plus breeding contract 5204.
The 2015 Dietary Guidelines for Americans: Adherence to Key Recommendations in Hispanic Females
Authors: Hannon Bridget , Villegas Elizabeth , Wiley Angela , Teran‐Garcia Margarita 
Published: June 2021

The Dietary Guidelines for Americans (DGAs) are released by the United States Department of Agriculture and Department of Health and Human Services to encourage consumption of a healthy eating pattern. Key recommendations of the 2015 DGAs include limiting saturated fats and sugars, and increasing consumption of a variety of fruits and vegetables, low‐fat dairy, and whole grains. The Healthy Eating Index (HEI) is commonly used as a measure of adherence, but the current HEI does not reflect the newest DGAs. Here, we propose a new HEI to assess adherence (HEI‐A) to the 2015 DGAs in a cohort of Hispanic females, a group disproportionately affected by obesity and related comorbidities.Fifty Hispanic females completed a semi‐quantitative food frequency questionnaire. Anthropometric measurements were also taken. Key recommendations from the 2015 DGAs for women were reviewed and an adherence index was derived from ten categories, each with a maximum score of ten. Total adherence was the sum of these 10 categories (max = 100). The HEI‐A is similar to the 2010 HEI, but reflects changes made in the 2015 DGAs.The average adherence score was 44.49 (SD: 14.47, 95% CI: 40.14–48.83). High fruit and vegetable intake was significantly associated with high adherence score (p=0.001). Low intake of saturated fat was significantly correlated with higher HEI‐A (r=0.71, p=0.03). The category with the greatest adherence was reduction of added sugars, with a mean score of 5.81 (SD: 2.94) out of 10. Adequate consumption of fruits and vegetables had the lowest mean of 2.89 (SD: 3.53). HEI‐A was not correlated with body mass index or waist circumference, and there were no significant differences between normal weight, overweight, or obese individuals.On average, participants were meeting fewer than half the recommendations of the 2015 Dietary Guidelines for Americans, regardless of weight status. Of note, the lowest adherence was in consumption of fruits and vegetables, but those individuals meeting this recommendation had overall greater HEI‐A scores. The DGAs are released to encourage healthy eating to prevent disease, and nutrition education tailored to Hispanics should continue to focus on emphasizing adherence to prevent obesity and related disease in this at‐risk population.Support or Funding InformationThis project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2015‐68001‐23248 from the USDA National Institute of Food and Agriculture.
Detection of Mature Plant miRNA in Different Biological Matrix: Importance of Internal Standard and Validation of the Method
Authors: Huang Haiqiu , Roh Jamin , Davis Cindy D. , Wang Thomas T.Y. 
Published: June 2021

MicroRNAs (miRNAs) ubiquitously exist in microorganisms, plants and animals, and appear to modulate a wide range of critical biological processes. However, no definitive conclusion has been reached regarding the uptake of exogenous dietary small RNAs into mammalian circulation and organs and cross‐kingdom regulation. One of the critical issues is our ability to assess and distinguish the origin of miRNAs. Although periodate oxidation has been used to differentiate mammalian and plant miRNAs, validation of treatment efficiency and inclusion of proper controls for this method were lacking in previous studies. This study aimed to address: 1) the efficiency of periodate treatment in plant or mammalian RNA matrices, and 2) the necessity of inclusion of internal controls. We designed and tested spike‐in synthetic miRNAs in various plant and mammalian matrices and showed that they can be used as control for the completion of periodate oxidation. We found that overloading the reaction system with high concentration of RNA resulted in incomplete oxidation of unmethylated miRNA. The abundant miRNAs from soy and corn were analyzed in the plasma, liver, and fecal samples of C57BL/6 mice fed with a corn and soy based chow diet using our improved methodology. We did not detect plant miRNAs in the mouse plasma or liver samples. In summary, an improved methodology was developed for plant miRNA detection that appears to work well in different sample matrices.Support or Funding InformationU.S. Department of Agriculture appropriated fund #8040‐51530‐056‐00D Office of Dietary Supplements Interagency Reimbursable Agreement #8040‐51530‐056‐20‐I
Impact of Usual Intake of Added Sugars on Nutrient Adequacy: NHANES 2009–2012
Authors: Nicklas Theresa 
Published: June 2021

The impact of usual intake (UI) of added sugars on nutrient adequacy in the US population was measured using NHANES 2009–2012 data of children (2–18 years; n=6,109) and adults (19+ years; n=10,697). Dietary intake data were obtained from 24‐hour dietary recall interviews using an Automated Multiple‐Pass Method. The National Cancer Institute (NCI) Method was used to estimate UI of added sugars and other nutrients. Individual UI of subjects 2–18 and 19+ years, were separated into six groups: 0<5, 5 to <10, 10 to <15, 15 to <20, 20 to <25, and ≥25% of energy as added sugars. Covariate adjusted regression coefficients were generated to examine the magnitude of the association between the percentages of the population below the EAR and added sugars intake. For most nutrients with an EAR (13/17 in children and 14/17 in adults) added sugars intake was not related (p>0.01) to nutrient adequacy. For those nutrients affected (primarily calcium (beta= 10.48; p=0.003) and vitamin E (beta=5.72; p= 0.003) for children and vitamins D (beta= 1.42; p= 0.001) and E (beta= 3.95; p= 0.008) for adults) the percentage of the population with inadequate intakes were high even in the lower added sugars groups. The point of inflection where micronutrient intakes appeared to be most apparent for these nutrients was between 20% – <25% of total energy from added sugars; regression analyses using four groups (0– <5, 5– <10, 10– <15, 15– <20 % energy as added sugars) did not show significant relationships with the nutrients studied. These results suggest that poor food choices independent, or only partially related to added sugars intake, may have a larger impact on nutrient adequacy rather than added sugars specifically. Future work might consider co‐modeling added sugars intake with energy intake using more sophisticated models becoming available.Support or Funding InformationUSDA/ARS through specific cooperative agreement 58‐6250‐6‐003 and from the USDA Hatch Project LAB 94209. Partial support was also received from the Corn Refiners Association.
Preterm Birth and Biomarkers of Environmental Enteric Dysfunction among Infants in Tanzania
Authors: Liu Enju , Manji Karim P , McDonald Christine M , Gosselin Kerri , Kisenge Rodrick , Fawzi Wafaie W , Gewirtz Andrew T , Duggan Christopher P 
Published: June 2021

ObjectiveTo examine the relationship between preterm birth and biomarkers of environmental enteric dysfunction (EED) among infants.MethodsWe performed a longitudinal study among a subset of Tanzanian children (n=590) who were enrolled in a micronutrient supplementation trial at age 6 weeks. Blood samples were collected at 6 weeks, 6 months, and 12 months to test for biomarkers of EED (antibodies to lipopolysaccharide (anti‐LPS) and flagellin (anti‐flagellin)). Longitudinal data analysis strategy (proc mixed in SAS) was used to examine the association between preterm birth and EED biomarkers. We adjusted for potential confounders including maternal age, maternal education, maternal middle arm circumference, daily food expenditure, household wealth, infant sex, and delivery by cesarean section. Tukey‐Kramer methods were used to adjust for multiple comparisons.Results56 (9.5%) infants were born preterm (defined as gestational age <37 weeks). Serum concentrations of all EED biomarkers increased over time (P trend<0.001). In multivariate analysis, at age 6 weeks, preterm infants had significantly higher serum concentrations of anti‐flagellin IgG (adjusted mean: 0.67 vs 0.49, p<0.01), anti‐flagellin IgA (0.45 vs 0.29, P<0.01), anti‐LPS IgG (1.05 vs. 0.78, P<0.01), anti‐LPS IgA (0.62 vs 0.42, p<0.01) compared to full‐term infants. At follow‐up (6 and 12 months of age), these differences did not persist (figure 1).ConclusionTanzanian infants born at < 37 weeks gestational age had significantly higher serum concentrations of four biomarkers of EED at 6 weeks of age, compared to full term infants. These differences did not persist later in infancy. Our data suggest that preterm infants may be uniquely susceptible to EED, and that this gastrointestinal dysfunction may have implications for growth and health outcomes in infancy and later. More efforts should be made to identify maternal, environmental, and dietary determinants of EED biomarkers from pregnancy to early childhood.Support or Funding InformationSupport for this effort was provided jointly by NIH (grants R01 HD048969 and K24 DK104676) and the Feed the Future Innovation Lab for Nutrition which is funded by the United States Agency for International Development (USAID)
Evaluation of Fluorescence Microsphere Immunoassay for Antibody Detection to Rift Valley Fever Nucleocapsid Protein and Glycoproteins
Authors: Ragan Izabela , Faburay Bonto , McVey D Scott , Richt Juergen A , Davis A Sally , Rowland Raymond R , Wilson William C 
Published: June 2021

Rift Valley Fever Virus (RVFV) is a zoonotic disease that infects ruminants including cattle, sheep, goats, camels and buffalo. There is a need to create a safe, rapid, and accurate assay that can simultaneously detect antibodies directed against multiple RVFV antigens. We describe the improvement and validation of a fluorescence microsphere immunoassay (FMIA) for the detection of IgG antibodies against the RVFV glycoproteins and the immunodominant nucleocapsid (N) protein. The purpose of this assay is to simultaneously screen and confirm sera from animals for positive or negative disease status. Well‐characterized vaccinated and experimentally infected sheep and calf sera were used for the evaluation of the assay. Recombinant viral proteins were produced then coupled to polystyrene magnetic beads for analysis using the Luminex MAGPIX system with xMAP technology. An unrelated viral antigen coupled control bead set was added to the reaction in order to account for non‐specific binding of antibodies to the antigen of the antigen‐bead complex. Median Fluorescence Intensity (MFI) results were converted to Sample/Positive ratios in order to standardize test results. Our results revealed highest MFI values for the detection of IgG antibodies against the RVFV N protein, indicating that this RVFV antigen could serve as a good candidate for a screening assay. The glycoprotein Gn target has been shown to differentiate vaccinated from non‐vaccinated animals in a candidate subunit vaccine formulation based on the RVFV Gn and Gc proteins. The FMIA demonstrated a high correlation (R2) of 0.91 when compared side by side with plaque reduction neutralization tests. The addition of further RVFV targets such as the non‐structural proteins NSs and NSm is currently under investigation. The results presented in this report demonstrate that FMIA provides a rapid and robust serological diagnostic tool for the detection of antibodies against RVFV. The targets developed in this assay will provide the basis for the development of a multiplex sero‐diagnostic assay system that can simultaneously screen for several ruminant diseases including transboundary animal diseases.Support or Funding InformationThis material is based upon work supported by the Kansas State NBAF Transition Funds, Kansas State University College of Veterinary Medicine, University of Pretoria Department of Veterinary Tropical Diseases, the USDA Agricultural Research project number 5430‐32000‐005‐00D and the U.S. Department of Homeland Security under Grant Award Number DHS‐2010‐ST‐061‐AG0001. The views and conclusions contained in this document are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the U.S. Department of Homeland Security.
Controlling Hydrogen Sulfide concentrations via PDMS microfluidics for endothelial cell culture
Authors: Christoforidis Theodore , Driver Tom , Rehman Jalees , Eddington David T 
Published: June 2021

BACKGROUNDHydrogen Sulfide (H2S), along with carbon monoxide and nitric oxide, has been found to be one of the cell gasotransmitters affecting vascular cell function, blood vessel growth as well as modulating the potassium‐ATP channels . Delivery of consistent hydrogen sulfide levels to study its physiological effects has been a challenge because of the rapid degradation of the molecule and this represents an obstacle in the field. Current cell culture studies are implemented with hydrogen sulfide concentrations that are not stable with time as the donors used degrade with time.PURPOSETo develop a system that would allow controllable hydrogen sulfide concentrations for cell cultures.METHODSWe developed a novel approach which delivers H2S via diffusion from microfluidic channels through a gas permeable membrane fabricated using PDMS soft lithography (Figure 1). The device consists of 3 different layers, the microfluidic channels, a 100μm thick PDMS membrane and a reservoir, where the cells can be seeded with appropriate amount of media. H2S molecules diffuse through the PDMS membrane to the cell culture reservoir. In this system, a defined mixture of sodium hydrogen sulfide (NaHS), used as a H2S donor, was injected through the microchannels using a syringe pump (Harvard Apparatus) . The detection of the H2S concentration was achieved using electrochemical sensors (Alphasense, Essex, UK). The electrical signal is acquired with a data acquisition system (NI‐9205, National Instruments, TX) using Labview.RESULTSIt is shown that H2S concentration is dependent on the concentration of the solution of NaHS and the flow rate of the solution in the microchannels. In this way different concentrations of H2S can be obtained in a controllable way. Our microfluidic approach enables the study of H2S in the range of 0 – 4.2 ppm (0–123 μM) which corresponds to the physiological concentrations in the low μM range (0.5–120 μM) (figure 2 & 3) .CONCLUSIONSStudying the impact of hydrogen sulfide in cell cultures is not yet well understood. We provide a method to control hydrogen sulfide for cell cultures. To our knowledge there is no other in vitro model that hydrogen sulfide concentration can be controlled.Support or Funding InformationNational Science Foundation 1253060, DTE.