bluuebunny/crossref_metadata_2025_split huggingface dataset:
A conservative isoleucine to leucine mutation causes major rearrangements and cold‐sensitivity in KlenTaq1 DNA polymerase (551.1)
Authors: Wu Eugene , Walsh Amanda , Materne Emma , Kornberg Emily , Zielinski Bryan , Barnes Wayne , Kermekchiev Milko 
Published: June 2021

Assembly of polymerase chain reactions at room temperature can sometimes lead to low yields or unintentional products due to mispriming. Mutation of isoleucine 707 to leucine in DNA polymerase I from Thermus aquaticus substantially decreases its activity at room temperature without compromising its ability to amplify DNA. To understand why a conservative change to the enzyme over 20Å from the active site can have a large impact on its activity at low temperature, we solved the X‐ray crystal structure of the large (5´‐to‐3´exonuclease‐deleted) fragment of Taq DNA polymerase containing the cold‐sensitive mutation (Cs3C KlenTaq1) in the ternary (E‐DNA‐ddNTP) and binary (E‐DNA) complexes. The Cs3C KlenTaq1 ternary complex was identical to the wild‐type in the closed conformation except for the mutation and a rotamer change in a nearby phenylalanine 749, suggesting that the enzyme should remain active. However, soaking out of the nucleotide substrate at low temperature results in an altered binary complex made possible by the rotamer change at F749 near the tip of the polymerase O‐helix. Surprisingly, two adenosines in the 5´‐template overhang fill the vacated active site by stacking with the primer strand, thereby blocking the active site at low temperature. Replacement of the two overhanging adenosines with pyrimidines substantially increased activity at room temperature, confirming the importance of base stacking. These results explain the cold sensitivity phenotype of the I707L mutation in KlenTaq1 and serve as an example of a large conformational change affected by a conservative mutation.Grant Funding Source: WMB supported by SBIR grant R44 GM6081002A1 and 1R21HG006291
Proteasome‐mediated degradation of TATA binding protein (TBP2) in Haloferax volcanii (556.1)
Authors: Fu Xian 
Published: June 2021

Gene transcription and proteolysis are two processes that have seemingly nothing in common: transcription is the first step in the life of any protein and proteolysis is last. However, we show that TATA binding protein (TBP2), one of the general transcription factors in Haloferax volcanii is degraded in proteasome‐mediated way. To be specific, TBP2 levels are increased by either chemical inhibition or genetic depletion of catalytic 20S core particles of proteasomes. In addition, TBP2 levels are higher in AAA+ ATPases proteasome‐activating nucleotidase (PAN) and cell division cycle 48 (cdc48) mutant strains compared with wild‐type strain suggesting TPB2 turnover by proteasome also requires AAA+ ATPases which closely related to the regulatory particle triple‐A ATPases (Rpt) of eukaryotic 26S proteasomes. Interestingly, like ubiquitin‐proteasome system in eukaryotic the recent described small ubiquitin‐like archaeal modifier proteins (SAMPs), its activating enzyme, E1 like UbaA, and the potential de‐ubiquitin enzyme (JAMM2) seem to be involved in TBP2 degradation based on the fact TBP2 level are much higher in those mutant strains than wild‐type strain, although evidences also suggest the presence of a SAMP‐independent pathway for TBP2 degradation. Overall, these findings first open a window for disclosing interactions among transcription initiation, SAMP and proteasome systems in archaea.Grant Funding Source: NIH R01 GM057498 and DOE DE‐FG02‐05ER15650
Peroxygenase and oxidase activities of dehaloperoxidase‐hemoglobin from Amphitrite ornata (580.1)
Authors: Ghiladi Reza , McCombs Nikolette 
Published: June 2021

The marine globin dehaloperoxidase‐hemoglobin (DHP) from Amphitrite ornata was found to catalyze the H2O2‐dependent oxidation of monohaloindoles, a previously unreported class of substrate for DHP. Using 5‐Br‐indole as a representative substrate, the major monooxygenated products were found to be 5‐Br‐2‐oxindole and 5‐Br‐3‐oxindolenine. Isotope labeling studies confirmed that the oxygen atom incorporated was derived exclusively from H2O2, indicative of a previously unreported peroxygenase activity for DHP. Peroxygenase activity could be initiated from either the ferric or oxyferrous states with equivalent substrate conversion and product distribution. It was found that 5‐Br‐3‐oxindole, a precursor of the product 5‐Br‐3‐oxindolenine, readily reduced the ferric enzyme to the oxyferrous state, demonstrating an unusual product‐driven reduction of the enzyme. As such, DHP returns to the globin‐active oxyferrous form after peroxygenase activity ceases. Reactivity with 5‐Br‐3‐oxindole in the absence of H2O2 also yielded 5,5’‐Br2‐indigo above the expected reaction stoichiometry under aerobic conditions, and O2‐concentration studies demonstrated dioxygen consumption. Non‐enzymatic and anaerobic controls both confirmed the requirements for DHP and molecular oxygen in the catalytic generation of 5,5’‐Br2‐indigo, and together suggest a novel oxidase activity for DHP.Grant Funding Source: Supported by the National Science Foundation CHE‐1150709
Mechanistic and computational studies on C‐N bond oxidation in D‐amino acids catalyzed by D‐arginine dehydrogenase Y53F and Y249F (584.4)
Authors: Gannavaram Swathi , Sirin Sarah , Gadda Giovanni 
Published: June 2021

Pseudomonas aeruginosa D‐arginine dehydrogenase (PaDADH) is a flavin dependent enzyme that can be classified as a member of D‐amino acid oxidase, sarcosine oxidase, and related enzyme families. It catalyzes the oxidation of D‐amino acids except D‐aspartate, D‐glutamate and glycine, to imino acids. Mechanistic details of C‐N bond oxidations of D‐amino acids by PaDADH are still elusive. Multiple mechanistic possibilities such as carbanion, hydride transfer and polar nucleophilic mechanisms could be considered. Previous work on PaDADH showed that NH and CH bond cleavages during amine oxidation are asynchronous with no further details available on the chemical mechanism. Here, we mutated two conserved active site residues Y53 and Y249 to phenylalanine in an attempt to reveal the mechanistic details of amine oxidation via pH profiles and deuterium isotope effects on rapid reaction kinetics. To further minimize mechanistic possibilities, QM/MM methodology was used to probe the relative local electrophilicity of key FAD sites within PaDADH active site. Our results are consistent with D‐amino acid oxidation occurring through the direct transfer of a hydride ion from the substrate to FAD without stabilization of reaction intermediates or FAD‐derived adducts, in which the active site tyrosines do not have a direct role in catalysis in PaDADH.Grant Funding Source: NSF MCB‐1121695 (G.G.) and a GSU Molecular Basis for Disease Fellowship (S.G.).
Human rhinovirus elicits differential cytokine response and signal transduction pathways in human macrophages as a function of viral receptor stimulation (609.2)
Authors: Hall David , Schuler Bryce , Schreiber Michael , Li LuYuan , Raugi Dana , Kingdon Megan , Smith Cosonya , Hameister Chelsea , Mokry Michal , Racaniello Vincent 
Published: June 2021

Major‐ and minor‐group rhinoviruses enter their host by binding to the cell surface molecules ICAM‐1 and LDL‐R, respectively, which are present on both macrophages and epithelial cells. Although epithelial cells are the primary site of productive HRV infection, previous studies have implicated macrophages in establishing the cytokine dysregulation that occurs during rhinovirus‐induced asthma exacerbations. Upon closer examination in human primary macrophages, differential signaling pathway activation was observed between major‐ and minor‐group rhinovirus upon initial HRV binding, indicating a discordant receptor‐dependent response to these rhinovirus types. Differential phosphorylation of kinases (p38, JNK, ERK5) and transcription factors (ATF‐2, CREB, CEBP‐beta) was observed between the major‐ and minor‐ group HRV treatments. Furthermore, major‐ and minor‐ group HRV elicited differential production of the asthma‐relevant cytokines CCL20, CCL2, and IL‐10. This is the first report of genetically similar viruses eliciting dissimilar cytokine release, transcription factor phosphorylation, and MAPK activation from macrophages. These results suggest that receptor dependence plays a role in establishing the inflammatory microenvironment initiated in part by monocytic‐lineage cells in the human airway upon exposure to rhinovirus.Grant Funding Source: Supported by the McNair Program, NIH R15 AI065505‐01A1,NSF 0521112, Excellence in Science Fund
Promoting concept driven teaching strategies in biochemistry and molecular biology through concept assessments: a regional workshop in Boston (618.16)
Authors: Fox Kristin , Roecklein‐Canfield Jennifer 
Published: June 2021

A workshop was held on January 11, 2014, at Simmons College in Boston, MA, to design assessments and learning strategies for concepts in Biochemistry and Molecular Biology Education. The workshop included faculty from a variety of institutions in the northeast. Before the meeting faculty filled out a survey indicating which of the recently‐published learning objectives, developed as part of the NSF‐RCN UBE grant referenced in the title, they were most interested in developing assessments for. Using this information, small groups of faculty were organized and each group worked to develop both an exam‐type question and a scoring rubric for the question. Once these had been developed, the groups re‐convened to list the knowledge from allied fields that is required to successfully answer the question. Finally, they worked together to design a student‐centered learning strategy that would prepare students to answer the question correctly. The assessments, rubrics and activities that were developed will be available for the community to use.Grant Funding Source: Supported by NSF Grant #DBI‐0957205
Undergraduate research ő a key to advancing interest in STEM (618.40)
Authors: Carroll Margaret , Skeete Dereck , Catapane Edward 
Published: June 2021

In 2006 Medgar Evers College received an NSF grant (0622197 of the DUE Program) designed to increase the number of students earning BS degrees in Biology and Environmental Science. The goals of our STEP into Science program were to: recruit new students and non‐STEM students into Biology or Environmental Sciences; improve retention by providing academic, financial and mentoring support; foster integration of research and technology to better equip majors to be successful applicants to graduate/professional programs; and increase the number of students graduating with BS degrees. We use peer recruiters to attract more high school, transfer, and non‐science college students into STEM majors and place emphasis on undergraduate research experiences to increase the quality and retention of science majors through their BS degree. Since the inception of the program, STEM enrollment more than doubled. Our program evaluations consistently showed that a key factor in our successful recruitment and retention was student involvement in undergraduate research activities. The number of majors actively engaged in research has risen more than 90% with a concurrent increase in student research presentations at scientific conference, and an 87% increase in the number of students receiving external research internships and travel awards to attend national conferences. STEM graduates have also increased and many of them are continuing on to Masters and Doctoral programs in STEM and hopefully will ultimately enter rewarding careers in the science enterprise.Grant Funding Source: NIH‐ 2R25GM06003, NSF‐ 0622197, NYSED‐ 0516041071
Digital exhaust: an analysis of students’ interactions with online learning tools (618.42)
Authors: Mueller Dustin , Offerdahl Erika , Boyer Jeff 
Published: June 2021

Learning management systems (LMS) such as Blackboard, Desire2Learn, and WebCT have become ubiquitous on college campuses. At a minimum, these platforms are an efficient way to provide access to course materials (e.g. course notes, syllabus, study guides) to all students. But the real power of LMS is in their potential to provide online tools to engage students outside of class in ways that support and reinforce the learning that occurs during class. When students interact with these online tools, they leave behind a digital “exhaust” that is collected in access logs. This clickstream data can be analyzed for patterns of student behavior from which we can make inferences about student learning. The overarching goal of this project was to analyze clickstream data associated with a large‐enrollment, introductory biochemistry course for science majors. Specifically, we analyzed students’ access logs related to their use of weekly Progress Checks. Progress checks are a series of online assessment tools released weekly to students that gauge their acquisition of key biochemical principles and skills as well as introducing them to upcoming material to be covered in class. We hypothesized that successful students would exhibit different clickstream patterns than non‐successful students. We gathered clickstream data, student performance data on in‐class activities, quizzes, and exams, incoming GPA, and pre/post assessment data. These data were analyzed for patterns of interaction that would help flag students who are not performing adequately within a course (i.e., an early warning system) to proactively target and support student learning.Grant Funding Source: Supported by the NSF
Best practices in direct nutrition education for low‐income audiences: educator characteristics, training, and monitoring (625.1)
Authors: MacKinnon Chloe , Auld Garry , Baker Susan 
Published: June 2021

The project objective was to identify best practices in nutrition education for low‐income audiences. Best practices are evidence‐based elements or strategies that have shown effectiveness in achieving outcomes. Utilizing best practices increases stakeholder confidence that programs will be effective in helping low‐income participants make positive nutrition‐related behavior changes. A seven‐member expert panel identified best practices criteria via telephone and electronic correspondence; the criteria were confirmed via literature review. Five criteria were identified as characteristics inherent to the success of the educator in community‐based nutrition education programs. These include expertise in both content and teaching methods, a thorough understanding of performance expectations, and the ability to relate to the target audience. Three criteria were identified for training and monitoring of educators throughout employment. Best practices criteria for educator characteristics, training, and monitoring are useful for program leaders at the state and local level and may promote consistency and efficacy within and across low‐income nutrition education programs.Grant Funding Source: Supported by the National Institute of Food and Agriculture no. 2012‐48757‐20337
Apples/apple products consumption is associated with a better nutrient intake profile, better diet quality, and lower risk of obesity in children (2‐18 yrs): National Health and Nutrition Examination Survey (NHANES) 2001‐2010 (626.16)
Authors: Nicklas Theresa , Rodriguez Gabriela , O'Neil Carol , Fulgoni Victor 
Published: June 2021

The association between apples/apple product (A/AP) consumption with nutrient intake, diet quality, and risk of obesity was examined in children using data from NHANES (2001‐2010). Consumers (n=3,482) were defined by consumption of any amount of apple, applesauce, or 100% apple juice using a 24‐hour diet recall. ANOVA (covariate adjusted) were conducted using appropriate sample weights. Logistic regression was used to determine odds ratios. Diet quality was calculated using the Healthy Eating Index‐2010 (HEI). Mean intake of total sugars (133±1 v 143±1 g/d), dietary fiber (12.2±0.1 v 15.0±0.2 g/d), magnesium (226±1 v 243±2 mg/d), and potassium (2149±16 v 2408±29 mg/d) was higher (p <0.01) in apple consumers than in non‐consumers. Intake of added sugars (21.3±0.2 v 17.8±0.3 tsp/d), SFA (26.3±1.5 v 24.6±0.2 g/d), and sodium (3172±20 v 3079±23 was lower (p <0.01) in A/AP consumers than non‐consumers. The total HEI score was higher (50.7±0.4 v 40.8±0.3) in A/AP consumers than non‐consumers. The percentage of obese children was lower among A/AP consumers (12.9%) compared to non‐consumers (17.6%). Risk of obesity was 25% lower in apple consumers but not significant among consumers of applesauce or 100% apple juice. Total consumption was associated with better nutrient intake, diet quality and weight parameters in children and consumption of A/AP should be encouraged.Grant Funding Source: Supported by: Dr. Pepper Snapple Group, Inc & USDA
Reduced IL‐10 production contributes to impaired NK cytotoxicity in old mice fed a high folic acid diet (638.3)
Authors: Sawaengsri Hathairat , Wang Junpeng , Reginaldo Christina , Smith Donald , Wu Dayong , Meydani Simin , Selhub Jacob , Paul Ligi 
Published: June 2021

Natural killer (NK) cells are important for immunity against malignant and virally infected cells. High folic acid intake, which may be linked with higher risk of colon cancer, was associated with lower NK activity in older women. To study the underlying mechanisms, we created a mouse model and found that aged mice fed a high folic acid (HFA) diet had reduced NK activity. In this study, we determined if expression of cytokines that are known to activate NK activity (IL‐10, IFN‐γ, TNF‐α) are affected by a HFA diet in aged mice.Female C57BL/6 mice (16 mo) were fed a control (1x RDA folic acid) or a HFA diet (20x RDA folic acid) for 3 months. Splenocytes were collected and stimulated with LPS for 24 h for ex vivo production of IL‐10, IFN‐γ, and TNF‐α determined by cytometric bead array.In HFA mice, IL‐10 production was 50.7% less than that of the control mice (P=0.028) but no differences were found in IFN‐γ and TNF‐α production. Since IL‐10 activates NK cells, we examined whether addition of IL‐10 to the splenocyte cultures would restore NK activity in the HFA group. We found that NK activity increased for both groups (P<0.01), but the HFA group had larger percent increase in NK activity as compared to control mice (P=0.058). These results indicate that IL‐10 restored the lower NK activity in aged mice fed a HFA diet.These findings suggest the decreased NK activity due to a high folic acid diet may in part be due to impaired IL‐10 secretion.Grant Funding Source: Supported by USDA contract # 51520‐008‐045 and HNRCA Pilot grant
Methods of sustaining dietary ketosis in Sprague‐Dawley rats (643.5)
Authors: Kesl Shannon , Poff Angela , Ward Nathan , Fiorelli Tina , Ari Csilla , D'Agostino Dominic 
Published: June 2021

The ketogenic diet (KD) has been successfully used to treat pediatric refractory epilepsy since the 1920s and is currently being investigated as an adjunct treatment for cancer and neurological disorders. The therapeutic efficacy of the ketogenic diet results from an elevation of blood ketones. Concerns regarding this treatment include low compliance due to restrictive diet and adverse effects on blood lipids. We hypothesized ketone precursor supplementation would elevate blood ketone levels to therapeutic ranges (2‐5mM) without need for dietary restriction. We tested the effects of the following ketone precursors on blood glucose, ketones, and lipids with a 28‐day dose escalation study in male Sprague‐Dawley rats: R,S‐1,3‐Butandiol (BD), a mineral (Na+/K+) salt of β‐hydroxybutyrate (βHB) (SO), medium chain triglyceride (MCT) oil (MO), SO+MO 1:1 mixture (SM), acetoacetate ketone ester (KE), and control (H2O) (n 蠅8). Days 1‐14, rats received a daily 5g/kg intragastric gavage, and days 15‐28 rats received a 10g/kg dose of their respective test substances. BD and KE rats were not increased to 10g/kg based on previous toxicology studies and were administered a 5g/kg dose for the duration of the study. Once weekly, whole blood samples (10 μl) were acquired for analysis of glucose and βHB at 0, 0.5, 1, 4, 8, and 12 hours after test substance administration, or until βHB returned to baseline. At day 1 and 28, 10 μL of whole blood were collected to measure triglycerides, total cholesterol, and HDL concentration. At days 1, 7, 14, 21 and 28 substances significantly decreased blood glucose and elevated βHB without dietary restriction. There were no significant elevations in the lipid panel for any of the substances.Grant Funding Source: Funded by the Office of Naval Research
Nutraceutical nanoemulsions: influence of physical states of β‐carotene (crystalized versus dissolved) on bioaccessibility (645.13)
Authors: Xia Ziyuan , McClements David , Xiao Hang 
Published: June 2021

β‐Carotene has a poor oral bioaccessibility in the gastrointestinal tract (GIT) because of its high hydrophobicity and melting point. We investigated the bioaccessibilities of solubilized and crystalline forms of β‐carotene using a simulated GIT model. Three systems were compared: (1) soluble β‐carotene in digestible nanoemulsion (d< 200nm); (2) crystalline β‐carotene added to digestible nanoemulsion (d< 200nm); (3) crystalline β‐carotene added to phosphate buffer saline (pH=7.0). Oil‐in‐water nanoemulsions were formed by high‐pressure homogenization using Tween 20 as emulsifier and corn oil as digestible carrier oil. They were then subject to simulated mouth, stomach and small intestine digestion. The rate and extent of free fatty acid production in the small intestine decreased in the order (2)>(1)>(3); whereas the β‐carotene bioaccessibility decreased in the order (1)>>(2)>(3). The low bioaccessibility (1.3%) of system (3) was attributed to the fact that there were few free fatty acids produced to form mixed micelles to solubilize β‐carotene. The bioaccessibility of system (2) was also relatively low (3.6%) compared to system (1) (42%), which suggests that crystalline β‐carotene is less bioaccessible than soluble β‐carotene. This study provides important information for choosing a better delivery condition for lipophilic bioactive components to achieve higher bioaccessibility.Grant Funding Source: Supported by USDA