bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chimeric apolipoproteins as novel plasma cholesterol lowering agents (1002.2)
Authors: Ibe Nnejiuwa , Lek Mark , Beck Wendy , Weers Paul , Narayanaswami Vasanthany 
Published: June 2021

Apolipoprotein E3 (apoE3, 299 residues, 34 kDa) and apolipoprotein A‐I (apoA‐I, 243 residues, 28 kDa) play significant roles in regulating plasma cholesterol levels, by virtue of their abilities to facilitate cholesterol uptake by binding the low density lipoprotein receptor (LDLr) and promoting cholesterol efflux from macrophages, respectively. We aim to generate a chimeric apolipoprotein by combining the LDLr binding region in the N‐terminal domain (NT) of apoE3 (residues 1‐191) with the cholesterol efflux‐promoting segment in the C‐terminal domain (CT) of apoA‐I (residues 181‐243). Overlap extension PCR was employed to fuse the nucleotide sequence encoding apoE3 NT with apoA‐I CT, followed by ligation into a pET20b(+) vector. The DNA sequence of the apoE3‐NT/apoA‐I‐CT chimera was verified. Overexpression of the chimera in E. coli yielded ~40 mg/L of purified protein. SDS‐PAGE and Western blot analyses confirmed the presence of a protein band of ~31 kDa, and MALDI‐TOF mass spectrometry revealed a mass of 30,977 Da (expected mass: 30,975 Da). Preliminary circular dichroism data of the chimera revealed α‐helical character, similar to apoE3 and apoA‐I. Further biophysical, spectroscopic and, lipid‐, LDLr‐binding and cholesterol efflux functional assays will be performed. The chimera has the potential to be used as a powerful cholesterol‐lowering agent to protect against cardiovascular disease.Grant Funding Source: Supported by the NIH #GM105561, #GM089564, and MARC grant T34GM008074‐22
Fluorinated aromatic amino acids distinguish side chain cation‐π interactions from membrane insertion (1002.5)
Authors: Roberts Mary , He Tao , Gao Jianmin 
Published: June 2021

Peripheral membrane proteins use a variety of motifs to bind transiently to membranes. For aromatic amino acids that are critical for membrane binding, two types of interaction can be envisioned: side chain insertion into the hydrocarbon milieu or formation of an amino acid π ‐ choline cation complex with phosphatidylcholine (PC). Distinguishing between these two can be difficult. However, site specific incorporation of fluorinated tyrosine or phenylalanine should differentiate these two binding modes. A fluorinated aromatic ring is more hydrophobic, favoring insertion, but the electrostatics of the ring have been changed such that the center is electropositive, destabilizing cation‐π complexes. A variant of S. aureus phosphatidylinositol‐specific phospholipase C that has an enhanced affinity for PC (N254Y/H258Y) was used to test this. If H258Y, a key residue in a helix that approaches the membrane surface, is replaced by either pentafluoro‐Phe or difluoro‐Tyr, binding affinity for PC‐rich membranes (measured by FCS) is lost. In contrast, when Phe249, a residue in a loop thought to insert into membranes, is replaced by pentafluoro‐Phe, the binding affinity is increased. Mutated Tyr that do not participate in membrane binding show no change in membrane binding affinities of the protein. This strategy offers the ability to test the role of Tyr and Phe in any peripheral membrane protein that can be expressed in E. coli.Grant Funding Source: NIH GM60418
Mannose‐based inhibitors of Escherichia coli adhesion: design and applications (1007.2)
Authors: Bouckaert Julie 
Published: June 2021

Structure‐thermodynamic relationships in protein‐carbohydrate interactions provide important decision makers in the design and maturation of bacterial ant‐adhesives. Many interactions in our innate defense depend on the specific recognition, of a high enough density of a glycan sequence, to evoke immune reaction. Bacteria hijack glycan receptors that are reminiscent of a physiological state of a mamalian cell that indicates a suitable habitat for the completion of their pathogenic cycle. The interruption of this process is envisioned using anti‐adhesives that selectively block bacterial adhesion but do not interfere with the immune system.The understanding of the biophysical basis of host‐pathogen interactions to empower intervention can be studied using purified adhesins and synthetic multivalent inhibitors. We measured affinities and stoichiometries using surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). The geometry of a multivalent complex was determined using small angle X‐ray scattering (SAXS) and was found to agree with the stoichiometry and molecular size derived from ITC and DLS data respectively. Stunningly, a correlation could be found between the dispersion part of interaction energies (quantum chemical calculations), ITC‐derived entropies and crystal structure electron densities of inhibitors bound to FimH.Grant Funding Source: Supported by ANR‐12‐BSV5‐0016‐01
Characterization of an O‐GlcNAc‐binding protein (1007.3)
Authors: Groves Jennifer , Zachara Natasha 
Published: June 2021

O‐linked‐β‐N‐acetylglucosamine (O‐GlcNAc) is a dynamic post‐translational modification of nuclear, cytoplasmic, and mitochondrial proteins. While O‐GlcNAc has been demonstrated to modulate numerous cellular processes, the molecular mechanisms by which O‐GlcNAc mediates protein function are not well defined. One mechanism by which O‐GlcNAc may regulate protein function is by promoting protein‐protein interactions. In this study we have investigated whether the intracellular hyaluronan‐binding protein p32 binds proteins in an O‐GlcNAc‐dependent manner. Using ELISA and dot blotting techniques we have demonstrated that p32 binds GlcNAc‐conjugates preferentially to other sugar‐conjugates in vitro. Suggesting that p32 interacts with O‐GlcNAc in vivo, p32 enriches O‐GlcNAc‐modified proteins similar to the O‐GlcNAc‐specific antibody RL2. Finally, our data demonstrate that overexpression of p32 fused to the biotin ligase BirA results in a biotinylation pattern that can be modulated by manipulating O‐GlcNAc levels. Preliminary studies suggest that one candidate protein bound by p32 in an O‐GlcNAc‐dependent manner is the O‐GlcNAc transferase (OGT), the enzyme responsible for the addition of O‐GlcNAc. Taken together, these data suggest that p32 may contain an O‐GlcNAc‐binding motif that dictates its ability to interact with a subset of intracellular proteins such as OGT.Grant Funding Source: Supported by the National Heart, Lung, and Blood Institute (P01HL107153)
Deciphering the dynamic regulation of phosphoinositide 3‐kinases downstream of G‐protein coupled receptors and receptor tyrosine kinases (1008.1)
Authors: Burke John , Vadas Oscar , Perisic Olga , Williams Roger L 
Published: June 2021

>Class I phosphoinositide 3 kinases (PI3K) are activated downstream of G protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs), mediating a number of physiological, and pathophysiological processes including inflammation, and tumour growth. We have determined the molecular basis for the activation of PI3Ks downstream of GPCRs and RTKs by applying a synthesis of hydrogen deuterium exchange mass spectrometry (HDX‐MS), X‐ray crystallography and biochemical/cellular assays. HDX‐MS provides information on the conformation and solvent accessibility of protein amide hydrogens, and has allowed us to interrogate the structural dynamics of these protein complexes on a membrane surface.The class I PI3Ks are composed of four different catalytic isoforms (p110α, p110β, p110δ, and p110γ), and only the p110β and p110γ isoforms can be activated directly by Gβγ subunits derived from GPCRs. However, this interaction only occurs on a membrane surface. By biochemically reconstituting PI3K with Gβγ on a membrane surface and carrying out HDX‐MS experiments we were able to determine the molecular interface of both p110β and p110γ with Gβγ subunits. Intriguingly we find that membrane binding causes conformational changes at the Gβγ interface, revealing a key dynamic role for membrane binding in priming activation downstream of GPCRs. Mutations that disrupted the Gβγ interface prevented oncogenic transformation, revealing a key role of GPCR signalling in mediating oncogenic transformation through PI3Ks.
UV irradiation/cold shock‐induced NOS2 expression for causing nuclear bubbling is WWOX and p53 dependent (1010.11)
Authors: Chang Nan‐Shan , Chen Szu‐Jung , Lin Pei‐Wen 
Published: June 2021

When a cell receives UV irradiation and cold shock, an untypical apoptosis occurs rapidly in less than 5‐20 min. We determined here that the UV energy activates cytosolic NOS2 (Nitric oxide synthase 2) for generating nitric oxide (NO)‐containing gas, which causes nuclear pore enlargement and a single large‐size bubble formation, as determined by electron microscopy (thus designated bubbling). Unlike typical apoptosis, no DNA fragmentation, nuclear condensation, membrane blebbing, and flip over of phosphatidylserine (PS) on cell membrane was observed. UV irradiation activated tumor suppressor p53 with phosphorylation at Ser46 and WWOX at Tyr33. Intriguingly, cold shock, but not UV, activates antiapoptotic TRAF2 (TNF‐α receptor‐associated factor 2) and downstream NFκB (nuclear factor κB). TRAF2 is abundant in the normal skin, and is significantly decreased in frostbitten skin tissues. In contrast, the expression of tumor suppressor WWOX is greatly upregulated in frostbitten skin. Mechanistically, UV/cold shock induced WWOX release from the WWOX/TRAF2 complex in 30 minutes, and the released WWOX bound p53 for relocating to the nucleus and caused cell death. Both dominant negative p53 and WWOX abolished the effect of UV/cold shock. Also, the UV/cold shock effect was blocked in Wwox‐/‐ MEF cells and in cells transiently overexpressed with TRAF2. Taken together, UV energy activates cellular NOS2 to generate nitric oxide‐containing gas in the nucleus, and causes cell damage in a WWOX/p53‐dependent manner. (Research supported by NSC and NHRI, Taiwan, and DoD, USA)Grant Funding Source: national science council; national health research institute
Anti‐apoptotic mechanism(s) of angiotensin 1‐7/mas in alveolar epithelial cell survival (1010.3)
Authors: Gopallawa Indiwari , Uhal Bruce 
Published: June 2021

Earlier work showed that autocrine synthesis of angiotensin II (ANGII) and c‐Jun‐N‐terminal kinase phosphorylation (pJNK) are required events in alveolar epithelial cell (AEC) apoptosis which is linked to severe lung diseases. Angiotensin 1‐7 (ANG 1‐7) prevents AEC apoptosis which is induced by locally synthesized ANG II. However the exact inhibitory mechanisms of ANG 1‐7/mas are currently unclear in AECs. The objective of the current research is to identify molecular mechanism(s) by which ANG1‐7 promotes AEC survival. We hypothesized that ANG 1‐7 activates a map kinase phosphatase (MKP) and maintains low pJNK levels thereby preventing apoptosis. To evaluate whether adding ANG 1‐7 acts through MKP‐2, freshly prepared ANG 1‐7 (0.1µM) in serum free media was added to a mouse lung epithelial cell line (MLE‐12). Cell lysates were run on 10% SDS‐PAGE to detect MKP‐2 protein by Western blotting. Current preliminary data suggests that ANG 1‐7 increased MKP‐2 protein (p=0.0286). Further, blockade of endogenous ANG 1‐7 with the mas receptor antagonist A779 reduced MKP‐2 levels (p=0.0014). Moreover data shows that inhibition of protein kinase A (PKA) in MLE‐12s induced pJNK and hence may be involved in promoting AEC survival. The results from these studies suggest that MKP‐2 is a key protein in the ANG 1‐7/mas pathway, and may provide insights to potential therapeutic strategies for lung diseases that involve AEC apoptosis.Grant Funding Source: Supported by HL‐45136
Expression in yeast of the metacaspase scp3 from the fungus Schizophyllum commune (1010.8)
Authors: Kaminski Anne , Fox Kristin 
Published: June 2021

Caspase homologs, metacaspases, are found in fungi, protists, and protozoa and are involved in programmed cell death‐like behavior. There is also evidence that metacaspases are additionally involved in proteostasis and cell cycle regulation. Metacaspases contain a cysteine histidine catalytic diad and cleave after lysine and arginine. Some metacaspases are activated by calcium and autocatalytic processing occurs in several, but is not necessary for activity.Five metacaspases, named scp1‐5, have been identified from the fungus Schizophyllum commune with the goal of expressing and characterizing them. Previous attempts to express these metacaspases in E. coli have resulted in either low amounts of protein or protein that forms inclusion bodies. The yeast Saccharomyces cerevisiae is often used to express eukaryotic proteins and should allow the production of large amounts of soluble protein. Therefore, the expression of these metacaspases in yeast is being investigated as an alternative to expression in E. coli.The scp3 gene has been successfully cloned from S. commune and inserted into the vector pYES2.1/V5‐His‐TOPO (Life Technologies). The vector containing scp3 was transformed into S. cerevisiae. Expression has been attempted by induction with galactose through the GAL1 promoter in the vector. Scp3 will be detected by western blotting using a V5 antibody (Invitrogen).
Effects of heparin and heparin‐binding growth factor on human aortic adventitial fibroblasts (1012.4)
Authors: Kalle Fanta , Akins Robert 
Published: June 2021

Cardiovascular diseases are primary treated using bypass graft surgery and angioplasty; unfortunately, these procedures often fail due to maladaptive tissue remodeling leading to stenosis, fibrosis, and vessel failure. Methods to decrease failure rates are needed. We are developing instructive biomaterials for placement along the abluminal surface of at‐risk vessels to provide mechanical support and to deliver bioactive molecules and/or stem cells to help attenuate maladaptive responses, encourage healing, and improve clinical outcomes. The present study examines the effects of critical biomaterial components including basic fibroblast growth factor (FGF2), low‐molecular weight heparin (LMWH) and desulfated LMWH on vascular cells.Cell morphology, proliferation, and viability are assessed using fluorescence microscopy. Image Pro and SPSS software packages are used to acquire and analyze data. Data indicate that FGF2 and LMWH affect human aortic adventitial fibroblast (AoAF) phenotype. The presence of FGF2 significantly increased proliferation rates and the appearance of filopodia; whereas, the absence of FGF2 was associated with an increase in the prevalence of lamellipodia and stress fibers. Increasing LMWH doses correlated with decreased AoAF proliferation and alterations in cell morphology. Desulfated LMWH did not alter FGF2‐mediated effects on cell phenotype.These data suggest that the biomaterial components LMWH and FGF2 can influence the phenotype of cardiovascular cells like AoAFs. Although in vivo, LMWH is thought to potentiate FGF2‐induced effects, in our system, LMWH significantly decreased AoAF proliferation, particularly in the absence of FGF2. The sulfation state of LMWH was critical to these effects. Further studies to determine the mechanisms accounting for these effects and to establish combined effects on cell phenotype will be needed to enable development of injectable formulations that may be useful in improving clinical outcome.Grant Funding Source: supported by the Delaware INBRE program with a grant from the National Institute of General Medical
Applying a chemical genetic approach to inositol polyphosphate signaling (1013.10)
Authors: Bratz Jeffrey , Dul Barbara , Fiedler Dorothea 
Published: June 2021

Signal transduction is a key area of study as we try to understand habitual cell physiology, and also discover new targets for amelioration of disease symptoms. Inositol polyphosphates, IPs, are small molecule messengers that play a critical role in cellular signal transduction pathways.Several highly phosphorylated IPs, such as 5‐diphosphoinositol pentakisphosphate, (5‐PP‐IP5) have been linked to insulin signaling in mammalian cells and maintaining overall energy balance in yeast. While these phenotypes are exciting, the field has been limited by traditional genetic techniques, and the exact mechanism of action by IPs has remained elusive.Through the development of a chemical genetic technique largely applied to protein kinases, we have engineered analog sensitive kinases responsible for the metabolism of IPs in yeast. We have exhibited the functionality of these enzymes, while simultaneously displaying the ability to specifically and temporally inhibit their kinase activity in both in‐vitro and in‐vivo assays. We plan to use this system in conjunction with metabolomics to further elucidate the role that these intriguing small molecules play in signal transduction pathways.Grant Funding Source: NIH
Nucleotide regulation of soluble guanylate cyclase (1013.14)
Authors: Surmeli Nur , Marletta Michael 
Published: June 2021

The interaction of the enzyme soluble guanylate cyclase (sGC) with nitric oxide (NO) mediates numerous physiological and pathological processes. NO production regulates smooth muscle relaxation, myocardial function, perfusion and platelet aggregation. NO signaling engages the second messenger cyclic guanosine monophosphate (cGMP) synthesized by sGC. Due to the critical importance of cGMP in many cellular processes, sGC activity is tightly regulated by NO, ATP and GTP. The mechanistic details of sGC regulation by these molecules are not well understood. Nucleotides influence sGC activity via a poorly characterized allosteric site. The catalytic domain of sGC has two putative nucleotide binding sites. One of these sites lacks an aspartate residue that is essential for catalysis, and is denoted as the pseudosymmetric site. The role of pseudosymmetric site on nucleotide regulation was investigated by site‐point mutations at this site. Kinetic analysis of the pseudosymmetric site mutant at various NO states has shown that pseudosymmetric site is involved in nucleotide regulation of sGC, and the effect of nucleotides is also dependent on the NO binding state of the enzyme.Grant Funding Source: American Heart Association
Dynamics of dining in the groups of people (1019.17)
Authors: Sigirci Ozge , Wansink Brian 
Published: June 2021

Objective: In an all‐you‐can‐eat (AYCE) buffet, how does diners’ eating and taste orientations change based on the gender of people they are dining with? Methods: The consumption amounts and taste evaluations of diners eating at an AYCE restaurant were taken along with other measures of behavior and self‐perceptions. The differences between males sitting in groups of females and males sitting in groups of males vs. females sitting in groups of males and females sitting in groups of females are analyzed using T‐Test.Results: Males who dine with females eat more pizza and salad than males who dine with males (p<0.05); on the other hand, females who dine with males eat more pizza but less salad than females who dine with females. Males dining with females find the meal and pizza tastier and more enjoyable than males dining with males, whereas females dining with males find the meal and pizza less tasty and less enjoyable than females dining with females. Moreover, females dining with males think they ate more than they should have and felt rushed than females dining with females (p<0.05).Conclusions: Dining in a group of people with same sex vs. mixed sex influences diners’ eating behaviors. Although eating with females lead males to eat more, they also eat more of healthy options such as salad and enjoy the meal more. But eating with males lead females to eat more and choose more unhealthy options, and enjoy the dinner less. A diner wanting a more enjoyable and perhaps a healthier experience might want to realize that eating in groups and eating with males or females will affect and alter the eating behavior.
Evaluation of the effectiveness of a cafeteria‐based behavioral economics intervention designed to improve student intake of reimbursable meal components (1019.18)
Authors: Golub Haleigh , Gunther Carolyn , Kennel Julie 
Published: June 2021

Behavioral economics (BE), the study of the effects of psychological factors on decision‐making, can be used in school cafeterias to influence student food choice and consumption. This study aimed to determine the impact of a cafeteria‐based BE intervention on student intake of meal components (entrée (grain/protein), vegetable, fruit, milk) and associated key nutrients (eg, fiber). Based on an assessment of cafeteria environments using The Smarter Lunchrooms Movement protocol, a BE intervention (identical across sites) was implemented in 4 elementary schools. A process evaluation checklist measured fidelity of intervention. Plate waste, an indirect indicator of dietary intake, was estimated 3 times before and after the intervention by scoring each meal component on a 5‐point scale from 0 (all item eaten) to 4 (all item wasted). Estimated nutrient intakes were calculated by comparing plate waste data against nutrient content of food items per serving size. Of the 637 students’ trays surveyed from the 4 schools at baseline, 21.8% and 44.5% of students ate all or none of the entrée, respectively; 68.7% ate less than 1/4 of the vegetable; and 60.3% ate 3/4 or all of the fruit. Only 13.7% of students chose unflavored milk over flavored varieties. Findings from this study will determine the effectiveness of a BE intervention designed to increase student intake of reimbursable meal components and key nutrients in schools.Grant Funding Source: Supported by the Ohio Department of Education Team Nutrition grant
Purchasing behaviors of individuals who shop at corner stores (1019.6)
Authors: Craven Tara , Swanson Ann , Young Taylor , Martin Donna , Gibson Cheryl 
Published: June 2021

Background. The Jackson County MO Healthy Corner Store Initiative is designed to work with small retail establishments to increase healthy food availability where residents have limited access to full line grocery stores. As part of this initiative, we assessed the purchasing habits of individuals who frequent one of four neighborhood corner stores. Method. Intercept surveys were administered during 28 two‐hour sessions and 2 four‐hour sessions. Survey sessions were held early morning, mid‐day and late afternoon over a 2‐week period. Participants were asked to indicate age, gender, transportation mode, food purchase frequency, typical items purchased, perceived healthiness of food items available, what influenced purchase decisions, and what items they purchased that day. Results. 446 surveys were completed. Most participants were greater than 18 years old (88%), male (61%) and drove a car or truck to the store. About 36% reported visiting corner stores daily. Most participants purchased snack items (59%) and felt that available food items were somewhat healthy (38%). Chips (17%), regular soda (25%), alcohol (15%) and tobacco (14%) were common items purchased. Almost 50% of participants reported to be very interested in healthier food options while about 23% felt that prices of healthy food items were comparable to other stores. 44% indicated variety would be the most important factor when deciding to purchase healthy foods. Discussion. Intercept survey results provided insight about buying habits and what changes might be possible to increase healthier food purchases. Education about healthy food choices will be an important factor when implementing the Healthy Corner Store Initiative.Grant Funding Source: Supported by CDC