bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ras Residue Y71 Promotes Flexibility of Switch I and Switch II
Authors: Guterres Hugo , Mattos Carla 
Published: June 2021

Ras is a small GTPase of 21 KDa that switches between GTP‐bound active state and GDP‐bound inactive state. Structural studies based on X‐ray crystallography and NMR have shown that in its active GTP‐bound state, Ras can adopt multiple conformations, with the switch II R‐(reactive) state and T‐(tardy) state, and switch I state 1, and state 2 being among the most well studied. Ras must be in state 2 to interact with effector proteins and in the R‐state to be catalytically competent. Through accelerated MD (molecular dynamics) simulation analysis of Ras, residue Y71 on switch II stands out as one of the most flexible side chains, spanning a conformational range sufficiently large to interact with Y96 on helix 3 on one side of switch II, and with E37 on switch I. In addition, this dynamic behavior of Y71 tends to accompany R and T state conformational transition. These observations lead to further study of the role of residue Y71 in Ras. Here we present a crystal structure of H‐RasY71A solved to 1.81 Å resolution and intrinsic hydrolysis rates for H‐RasY71A and K‐RasY71F, relative to their wild type counterparts. In addition, we study the differences in dynamics of these proteins using classical MD simulation. The T‐state conformation is stabilized in the crystal structure of H‐RasY71A, with fewer H‐bond networks than the wild type connecting helix 3 to switch II. MD simulations show that switch I and switch II are more rigid in both H‐RasY71A and K‐RasY71F mutants relative to the wild type H‐Ras. Interestingly, intrinsic hydrolysis assays gives a higher rate constant for H‐RasY71A than for wild type, while for K‐RasY71F the rate constant is smaller. Overall, our results suggest that residue Y71 plays a significant role in promoting flexibility of switches I and II and that it plays an important role in the conformational transition between T and R states important in promoting intrinsic hydrolysis of GTP on Ras.Support or Funding InformationNSF MCB‐1517295
Engineering a Tunnel to a More Versatile Lipoic Acid Ligase
Authors: Schmitz Benjamin , Konkolewicz Dominik , Page Richard C 
Published: June 2021

Protein‐polymer conjugates have become a rapidly expanding area of research. As a result of broad utility in a range of disciplines, protein‐polymer conjugates can now be applied to therapeutics, biofuels, and even protein tags. Grafting‐to and Grafting‐from methods have been successful in creating these conjugates but have limitations on the specificity of attachment (Lele et al. 2005). Lipoic acid ligase (LplA) has been demonstrated to be convenient tool for attaching small molecule precursors that can enable subsequent attachment of a polymer to specific sites of target proteins containing a lipoic acid ligase acceptor peptide (LAP) sequence. Studies on LplA have demonstrated that mutation of tryptophan 37 to valine allows LplA to accommodate 10‐azidodecanoic acid (Plaks et al., 2015). Paired with an alkyne‐bearing polymer this enables the use of click chemistry to attach a polymer to the protein‐ligated 10‐azidodecanoic acid. While useful, attachment of a pre‐clicked polymer would enable for more rapid exploration of protein‐polymer conjugates with a wide library of polymers. In this study, we describe structure‐guided, activity‐based efforts to engineer a tunnel within LplA to further open the binding pocket and allow LplA to accept pre‐clicked polymers.Support or Funding InformationThe authors acknowledge financial support from the US National Science Foundation (Award No. MCB 1552113 to RCP), the Burroughs Wellcome Fund (Award No. 1014031 to RCP), and institutional support from Miami University through the Robert H. and Nancy J. Blayney Professorship (to RCP).
Regulation of Mitotic Microtubule Dynamic Instability in Monopolar Spindles by Bundling and Kinetochore Attachment
Authors: Gergely Zachary R , Flynn Patrick J , Montes Salvador , McIntosh J. Richard , Betterton M. D. 
Published: June 2021

ObjectiveThe dynamics of mitotic microtubules are important for mitotic spindle assembly and chromosome segregation, but descriptive parameters have proved challenging to measure directly. We are studying the dynamic instability of single and bundled mitotic microtubules (MTs) that originate from the spindle pole bodies in fission yeasts. We marked the α‐tubulin with an m‐Cherry tag expressed at low levels to minimally perturb MT dynamics and labeled the kinetochores with a GFP tag. Using a temperature sensitive kinesin‐5 allele (cut7–24) at restrictive temperature (37°C) in which bipolar spindle assembly is blocked, we observe monopolar spindles whose dynamic MTs can be imaged directly.Results and ConclusionsThis persistent monopolar spindle allows the measurement of growth, shrinkage, catastrophe, and rescue rates for both single and bundled MTs. The kinetochores are dynamic and appear to move along bundled MTs allowing us to study correlated dynamics of the MTs and kinetochores. We have compared MT dynamics and bundling in this strain with others that contain additional perturbations to proteins that affect MT dynamics and bundling. To quantify these parameters, we have authored software to automate detection and measurement of MT and kinetochore dynamics and their changes over time in monopolar spindles. To quantify the rate of tubulin turnover in this system, we have performed photobleaching experiments in which we detect the substitution of unbleached tubulin dimers for bleached ones using fluorescence recovery (FRAP).Support or Funding InformationThis work was supported by NSF grants DMR‐1551095 to MDB, and NIH grants K25 GM110486 to MDB and R01 GM033787 to JRM.
Identifying the Expression Patterns of xCT in Zebrafish to Determine its Role in Neuroregeneration
Authors: Solorzano Marissa A , Ladd Nicole A , Franz Kevin C , Da Silva Christopher O , Degnan Shannon M , Chase Leah A , Putzke Aaron P , Krueger Brent P 
Published: June 2021

System xc− is a heterodimeric amino acid transporter comprised of a transmembrane light chain unit, xCT, and an extracellular heavy chain unit, 4F2HC. System xc− has been shown to exchange intracellular glutamate for extracellular cystine, which is then reduced within the cell to cysteine, the limiting reagent for glutathione production. Glutathione is a reducing agent that is important in reducing oxidative stress, which untreated can trigger cell death. It has been shown that system xc− is strongly expressed in the central nervous system, particularly in neuroprotective cells such as astrocytes and microglia. It is believed the reduction of oxidative stress in the environment of neurons and neuroprotective cells is critical in processes such as neuroregeneration. The current focus of this study is to qualitatively determine the expression patterns of the xCT gene in zebrafish embryos using in situ hybridization; preliminary results will be presented. This is in preparation for exploring the role that xCT plays in neuroregeneration using laser ablation to mimic spinal cord injury in vivo.Support or Funding InformationNational Science Foundation award 1058981.
Activation of Akt increases Cell Surface Expression of System xc−
Authors: Versluis Philip , Gibson Amanda , Schmidt Mackenzie , Smith Daniel , Chase Leah 
Published: June 2021

System xc− is a heterodimeric plasma membrane transporter involved in the exchange of intracellular glutamate for extracellular cystine. As such, this transporter plays a critical role in the production of the antioxidant glutathione. Previous studies in our lab have demonstrated that there is an increase in cell surface expression within ten minutes of exposure to H2O2 in confluent U138MG human glioma cells. The study described herein sought to begin to characterize the mechanism by which H2O2 regulates the trafficking of xCT. We hypothesized that Akt signaling is necessary for H2O2‐mediated trafficking of of xCT. A significant increase in Akt phosphorylation was observed in U138MG cells following ten‐minute exposure to 3 mM H2O2 compared to vehicle‐treated cells using western blot analysis. Treatment with the Akt inhibitor 10‐DEBC (2.5μM) for 30 minutes prior to and during H2O2 exposure resulted in a decrease in H2O2‐induced phosphorylation of Akt at Ser473. Similar inhibition of Akt phosphorylation at Thr308 was observed following treatment of cells with 1.0μM API‐2. Next, we used simultaneous treatment of cultured glioma cells with both inhibitors in the presence of H2O2 to determine if such treatment led to a reduction in the trafficking of endogenously expressed xCT to the plasma membrane. Our preliminary data suggests that Akt activation is necessary for H2O2‐induced trafficking xCT to the plasma membrane in cultured glioma cells. To determine if the regulation of xCT cell surface expression is not limited to human glioma cells where xCT is oven overexpressed, we also studied the role Akt plays in the trafficking of recombinant, transiently‐expressed xCT in COS‐7 cells. COS‐7 cells transfected with myc‐tagged xCT, 4F2HC and a constitutively active form of Akt showed higher levels of xCT localized to the membrane compared with cells transfected with a dominant negative form of Akt. Collectively, these data suggest that Akt is an important regulator of xCT cell surface expression.Support or Funding InformationThis work was supported by NSF‐RUI 0843564.
Relationship between Solid Food Introduction and Picky Eating in the STRONG Kids 2 Cohort
Authors: Cole Natasha Chong , Lee Soo‐Yeun , Donovan Sharon M. 
Published: June 2021

Picky eating is prevalent in toddlers and has been linked to infant feeding practices. Early introduction of solid food is associated with picky eating; however, the relationship with specific types of complementary food is unknown. The objective of this study was to examine associations between types of solid food introduced during infancy and picky eating behavior in children 18‐months of age. Participants were drawn from an ongoing STRONG Kids 2 birth cohort of mother‐infant dyads (current study n = 259, full sample n = 440). Mothers responded to survey questions about the type and amount of solid food introduced during infancy, the perception of their child's picky eating behaviors at 18‐months, and whether they breastfed or formula‐fed their child during the introduction to solids. Ordinal logistic regression (i.e., proportional odds model) was used to determine associations between types of solid food introduced and odds of picky eating, while controlling for child sex, race/ethnicity, age of introduction to solids, and mode of milk feeding. The introduction of sweets (e.g., ice cream and pudding), cereal, pureed fruit, and egg yolk were positively associated with parental perception of picky eating at 18‐months (all p<0.05). There was no association between parental perception of picky eating and the introduction of vegetables, meat, or dairy. Overall, our findings suggest that early exposure to various tastes and textures may influence the development of picky eating later in childhood. Further research is needed to determine the influence of the timing and type of solid food introduction on children's long‐term dietary variety and eating behavior.Support or Funding InformationSupported by National Institute of Food and Agriculture, U.S. Department of Agriculture, under award number 2011‐67001‐30101 and the Dairy Research Institute.
Culturally‐Relevant Diabetes Education Program Viewed Favorably by Rural‐Residing Latinos
Authors: Keane Michelle , Bahl Morgan , Francis Sarah L 
Published: June 2021

Rural‐residing Latinos are at high risk for diabetes and diabetes complications. Culturally‐relevant diabetes education is needed for this group. Latinos Living Well (LLW) was created using the Social Marketing Theory (SMT) to ensure it was audience‐centered and culturally‐sensitive for rural‐residing Latinos in a Midwest state. LLW is a four‐week diabetes education program focused on diet and exercise. The two‐hour classes include culturally‐relevant physical activity and cooking practicums, group activities and goal setting. Classes were taught in Spanish and delivered through Cooperative Extension. This study evaluated participant satisfaction and perceptions of LLW. A convenience sample of rural‐residing Latino adults ages 18+ years (n=120) were recruited through word of mouth, flyers, and radio ads; 76 completed the program (63.3% completion rate). Participants completed sociodemographic questions at Class 1 and a qualitative program evaluation at the end of Class 4. The majority of participants were first generation immigrants (n=63; 87.5%), females (n=73; 97.3%), ages 30–49 (n=34; 51.6%), with at least 9–12 years of education (n=56; 77.8%). A diabetes or pre‐diabetes diagnosis was not required for enrollment into LLW; however, two‐thirds (n=49; 65.3%) reported a history of diabetes. The evaluation asked about participants' perceptions of LLW and what lifestyle changes were made as a result of attending. Descriptive statistics analyzed sociodemographic and evaluation data. Nearly all (n=69, 94.5%) believed LLW helped them to make healthy lifestyle changes. These changes included preparing at least one class recipe at home (n=57, 79.2%) and being more physically active (n=58, 81.7%). The content was viewed as understandable (n=72, 98.6%). The majority (n=70, 95.8%) rated LLW as “excellent” or “good” and would recommend the program to a friend (n=72, 94.7%). Results suggest rural‐residing Latinos enjoyed LLW, thus indicating using culturally‐relevant examples and activities helped participants view the program favorably. Furthermore, it is likely participants will refer friends and family, be motivated to apply the information they learned and attend other health programming through Cooperative Extension.Support or Funding InformationThis research was funded by the United States Department of Agriculture, National Institute of Food and Agriculture Rural Health and Safety Education Grant, #2014‐46100‐22349
Development and Validation of a B.E.A.N. Survey for College Students
Authors: Sowers Morgan , Colby Sarah , Allison Chelsea , Zhou Wenjun 
Published: June 2021

BackgroundCollege students often do not consume the recommended amounts of beans and legumes. To develop interventions aimed at improving the intake of beans and legumes in the college population, more information is needed on the behaviors, environment, attitudes, and nutrition knowledge (B.E.A.N.) of college students regarding beans. Validated tools to assess these bean‐related constructs are currently lacking.ObjectivesThe objectives of this study are to 1). Gather preliminary qualitative information about bean‐related constructs (Phase I); and 2). Create a valid and reliable survey tool to measure the B.E.A.N. constructs with a larger sample of college students (Phase II).MethodsPhase I included cognitive interviews with university students (n = 30) to collect baseline data on the B.E.A.N. constructs. Phase II involved survey development and content validation with 5 content experts and face validation with 5 undergraduate students. A convenience sample of undergraduate students was surveyed to test internal structure and consistency (n = 355), establish convergent and discriminant evidence (n = 351), and measure test‐retest reliability (n = 242) of the developed tool.ResultsDevelopment and revisions in Phase I and content and face validation resulted in a 13‐question survey consisting of four primary constructs of behavior (4 items), environment (2 items), attitudes (5 items), and nutrition knowledge (2 items). In Phase II, factor analysis resulted in one factor of “bean consumption” being created from combining attitude and behavior questions, with factor loadings from .776 to .947. However, two environment and two knowledge questions were retained due to perceived relevancy. The internal consistency was α = 0.967 for the “bean consumption” construct and α = 0.962 for the entire survey. The knowledge construct was validated by those that had taken an introductory nutrition course having significantly higher mean knowledge scores than those that had not taken an introductory nutrition course (p < 0.001; 3.05 ± .89 vs. 2.30 ± 1.62, respectively). The test‐retest results ranged from r = .480 to .825 with all correlations being statistically significant (p < 0.010; 2‐tailed). The average total score for the pilot testing was 34.74 (± 27.27) out of a possible 100 points.ConclusionThis validated survey may be used in future research to examine the B.E.A.N. of college students regarding beans. This knowledge may be valuable in developing future effective health promotion interventions with college students.Support or Funding InformationThis project was supported by Agriculture and Food Research Initiative Grant no. 2014‐67001‐21851 from the USDA National Institute of Food and Agriculture, “Get Fruved:” A peer‐led, train‐the‐trainer social marketing intervention to increase fruit and vegetable intake and prevent childhood obesity – A2101.
Impact of long‐term dietary patterns and short‐term nutrient intake on the gut microbiota of children 4 to 8 years of age
Authors: Berding Kirsten , Donovan Sharon 
Published: June 2021

The gut microbiota is undergoing rapid changes in the first few years of life and diet plays a fundamental role in shaping the gut microbiota. It is commonly believed that by age 3 years, the microbiota composition resembles that of an adult. However, recent research shows differences in the microbiota composition between pre‐adolescence children and adults. Little is known about how diet influences the gut microbiota composition in young, healthy children. Herein, the effect of long‐term dietary patterns and short‐term dietary intake on longitudinal changes in microbiota composition were studied in children aged 4–8 years. A food frequency questionnaire, 3‐day food record and stool sample were collected for each participant (n=24) at baseline, 6‐weeks and 6‐months post‐baseline. Nutrient intake was analyzed using Nutrition Data System for Research (NDSR). Dietary patterns based on food groups were identified using principal component analysis. Bacterial genomic DNA was analyzed for total bacteria, Prevotella, Lactobacillus, and Roseburia by quantitative real‐time qPCR. Distinct microbial profiles were identified for three long‐term dietary patterns. Compared to the other two dietary patterns, a diet pattern characterized by a higher intake of fruits, vegetables, meat, fish, legumes, nuts and seeds, but also sugary drinks and desserts showed higher abundance of total bacteria. A diet pattern defined by a higher intake of whole grains and starchy vegetables was characterized by lower density of Lactobacillus and Roseburia, but higher density of Prevotella. Lastly, a diet pattern characterized by higher intake of refined grains, French fries and a lower intake in salty snacks was associated with lower abundance of total bacteria. Short‐term dietary fiber intake was not significantly correlated with bacterial abundance. Overall, there was no significant difference in the mean of the bacteria concentration between baseline and 6 weeks. However, bacterial abundance differed for some individuals between the two time points. In conclusion, long‐term dietary patterns influence the fecal microbiota of children aged 4 to 8 years. Analysis of 6 month samples will inform findings relative to the longitudinal stability of the gut microbiota. Future analysis includes microbiome analyses using next generation sequencing and assessment of short chain fatty acid concentrations.Support or Funding InformationUSDA Hatch Grant ILLU‐698‐311
Vitamin K, Vitamin D, and Lower Extremity Function: Results from the Osteoarthritis Initiative and Health, Aging and Body Composition Studies
Authors: Shea Kyla , Loeser Richard F , Kritchevsky Stephen B , Houston Denise K , McAlindon Timothy E , Booth Sarah L 
Published: June 2021

Knee osteoarthritis (OA) is the leading cause of impaired lower‐extremity function in older adults. Vitamin K insufficiency has been associated with greater structural knee OA progression, but studies associating vitamin K status with functional consequences of knee OA are lacking. The purpose of this study was to determine the association between vitamin K intake and lower‐extremity function in the Osteoarthritis Initiative (OAI), a prospective observational study of knee OA (n=4497, 58% female, 61±9 years old). Because the mechanisms underlying the role of vitamin K in joint health also implicate vitamin D, we additionally evaluated the association of combined vitamin K and vitamin D intake with lower extremity function. Dietary intakes were estimated using a food frequency questionnaire at baseline. Lower extremity function was assessed at baseline and annually over 4 years using the 20‐meter gait speed and time to complete 5 chair stands. Participants in the highest quartile of vitamin K intake had significantly faster 20‐meter gait speed and chair stand time over 4 years compared those in the lower quartiles (both p‐trend<0.001; analyzed using linear mixed models adjusted for demographics, co‐morbidities, diet and lifestyle characteristics). When vitamin K and D intakes were considered together, those in the highest quartiles of both vitamin K (≥229 mcg/d) and vitamin D (≥600 IU/d) intake had significantly faster gait speed (p<0.001) and chair stand time (p=0.002) over 4 years compared to participants in the highest quartile of either nutrient alone and compared to participants in the lowest quartile of both nutrients. We sought to replicate these findings, given their novelty, in the Healthy, Aging and Body Composition Knee OA sub‐study (n=1102, 60% female, 75±3 years old). Vitamin K and D status were assessed using circulating vitamin K and 25(OH)D at baseline and lower extremity function was assessed using the short physical performance battery (SPPB) and 20‐meter gait speed at baseline and over 4–5 years of follow‐up. Participants with both sufficient vitamin K (≥1.0 nM) and 25(OH)D (≥50nM) had significantly better SPPB scores and faster gait speed over follow‐up compared to those with sufficient levels of either nutrient alone and compared to those who were insufficient in both (p=0.001 and 0.005 respectively; adjusted for demographics, season, co‐morbidities, diet and lifestyle characteristics). These findings suggest vitamin K and vitamin D may be mutually beneficial to lower‐extremity function related to knee OA but need to be confirmed in clinical trials.Support or Funding InformationSupported by the USDA (Cooperative Agreement 58‐1950‐7‐707) and the NIH (grants K01AR063167, P30AG21332, R01AG028050, R01NR012459, contracts N01AG62101, N01AG62103, N01AG62106)