bluuebunny/crossref_metadata_2025 huggingface dataset:
Live‐cell imaging study on anti‐adipogenic and apoptotic effects of a polyphenolic resveratrol during adipocyte differentiation (959.19)
Authors: Ihara Hayato , Ogawa Mikako , Magata Yasuhiro
Published: June 2021

In the present study, using fluorescent live‐cell imaging system, we demonstrate an apoptotic effects of resveratrol, a polyphenolic compound present in grapes and other foods, on adipocyte differentiation. We hypothesize that resveratrol inhibits adipocyte differentiation through an induction of apoptosis in early‐stage cells of adipocyte conversion. Adipocyte conversion and induction of apoptosis of 3T3‐L1 cells were observed using fluorescent dyes, BODIPY and Alexa568‐conjugated annexin V, respectively. Time‐lapse cell imaging was performed using BioStation system (Nikon Tokyo), which consists of CO2 incubator incorporating fluorescent microscopy with CCD camera. Resveratrol treatments during differentiating stage induced apoptosis not in mature adipocytes but in early‐stage cells of adipocyte conversion, which resulted in a dramatic inhibition of adipogenesis. Even in differentiated 3T3‐L1 adipocytes (Day 6), mature adipocytes did not undergo apoptosis in response to 24 hr‐treatments of resveratrol, whereas newly differentiating cells did. Next, we tried to elucidate anti‐adipogenic effects of resveratrol in vivo, ob/ob mice were treated with resveratrol by daily oral administration (50 mg/kg B.W.). After 1‐month treatment, 10% body weight loss was observed in these obese mice. Furthermore, CT scan before and after resveratrol administration showed that there were no clear regional differences in the reduction of adipose tissue mass, but abdominal subcutaneous adipose tissue seem to be reduced by these treatments. These results suggest that resveratrol reduce adipose tissue mass in part through an induction of apoptosis in the early‐stage cells of adipocyte differentiation.
EU‐OPENSCREEN: novel chemical tool compounds for molecular biologists (972.3)
Authors: Stechmann Bahne
Published: June 2021

EU‐OPENSCREEN (www.eu‐openscreen.eu) is an academic European open‐access research infrastructure on the European roadmap with the aim to develop novel research ‘tool compounds’ (i.e. chemical inhibitors or activators of biological targets) for all areas of the Life Sciences (incl. molecular, cell, plant and microbiology; chemistry; pharmacology, early drug discovery etc.). EU‐OPENSCREEN supports all stages of a tool development project, incl. assay miniaturization, high‐throughput screening and chemical optimization of ‘hit’ compounds.

EU‐OPENSCREEN offers to external researchers access to its shared resources, incl. the latest screening technologies, chemistry services and a unique compound collection. Applications of biologists are welcome who have developed a robust assay and are interested in collaboratively developing a chemical ‘tool compound’ for their target‐of‐interest. Selected assays are screened against 100,000‐200,000 compounds, incl. confirmatory and counter screening, IC/EC50 determination, SAR (structure‐activity relationships) and QC of confirmed hit compounds.

Currently in its conceptualization phase, EU‐OPENSCREEN will start operations in late 2015, but it can already look back on a growing number of transnational activities: joint screening projects, exchange of local compound libraries and development of new design principles for its compound collection.
Characterization and complementation of a potential novel phenotype for Saccharomyces cerevisiae PHO13 (976.5)
Authors: Gehret Austin , Blake Kimbria , Edwards Jasmine , O'Handley Suzanne
Published: June 2021

PHO13 in Saccharomyces cerevisiae is a member of the p‐nitrophenylphosphatase (p‐NPPase) family of the Haloalkanoic Acid Dehalogenase (HAD) superfamily. While the physiological role of this enzyme remains debated, our lab has demonstrated PHO13 possesses specific activity towards 2‐phosphoglycolate. Phosphoglycolate phosphatases (PGPases) are required in photosynthetic organisms to recycle this metabolite, a by‐product of the Calvin Cycle, yet their presence in non‐photosynthetic organisms suggests 2‐phosphoglycolate is generated in other pathways. Regardless of its origin in yeast, 2‐phosphoglycolate must be catabolized due to its tendency to strongly inhibit triose phosphate isomerase. We have identified a potential growth phenotype for pho13‐Δ cells, a decrease in lag phase upon hyperosmotic (1M NaCl) shock compared to wild‐type cells. We hypothesize that the excess of 2‐phosphoglycolate in pho13‐Δ cells leads to an increase in glycerol production that confers a growth advantage to hyperosmotically stressed cells. Studies are currently underway to attempt complemention of this phenotype in pho13‐Δ cells by PHO13 and gph, a previously characterized PGPase from non‐photosynthetic E. coli. Though the pho13‐Δ phenotype is not a direct measure of PGPase activity, it could serve as a useful downstream indicator of physiological PGPase activity.
Affinity extraction of proteins from acidic environments (979.2)
Authors: Teichert Kristian , Stocks Bradley , Engen John
Published: June 2021

Affinity extraction of proteins from acidic environments Affinity purification is widely used to isolate an analyte of interest from a complex mixture, such as a cell lysate. The process involves immobilization of one ligand followed by incubation with a sample containing the binding partner. While most affinity purifications are performed at near‐physiological pH, there are situations where performing affinity extraction at low pH would be advantageous, including isolating proteins from acidic environments such as the stomach or acidic hot springs. The affinity pairs most often used for protein purification (e.g., antibody:antigen, GST‐tags:glutathione or immobilized metals:6xHis tags) have limited or abolished binding at low pH. Here we employed a published double mutant of streptavidin which is known to form a tight complex with biotin at low pH. A vector designed to incorporate an N‐terminal biotinylation sequence onto a protein was used to direct in vivo biotinylation of a selected lysine residue in several target proteins. This strategy has advantages over non‐specific in vitro, post‐purification biotinylation wherein multiple labels are added randomly and thereby cause disruption of key protein‐protein interactions. We demonstrate the utility of the mutant streptavidin:biotin system for extracting proteins of interest by characterizing the binding kinetics of this affinity pair in various solutions, pH’s and temperatures. Support: NIH R01‐GM101135

Grant Funding Source: Supported by NIH R01‐GM101135
Protein‐protein interactions between CIB1 and novel integrin binding partners: structural and thermodynamic basis of CIB1 promiscuity (979.5)
Authors: Freeman Thomas , Black Justin , Bray Holly , Dagliyan Onur , Wu Yi , Tripathy Ashutosh , Dokholyan Nikolay , Leisner Tina , Parise Leslie
Published: June 2021

The cytoplasmic protein CIB1 (calcium‐and‐integrin‐binding protein 1) has been shown to promote cancer cell growth by affecting the RAS/RAF/MEK/ERK and the PI3K/AKT pathways, and was previously identified as an αIIb binding partner that inhibits agonist‐induced activation of the platelet‐specific integrin, αIIbβ3. To better understand the nature of the protein‐protein interactions between CIB1 and other proteins, we characterized the interaction between CIB1 and several α‐integrins. A sequence alignment of all α‐integrin CTs revealed that key residues in the CIB1 binding site on αIIb are well‐conserved, and was used to delineate a consensus binding site (I/L‐x‐x‐x‐L/M‐W/Y‐K‐x‐G‐F‐F). A hydrophobic binding pocket on CIB1 was shown to be the major binding site for multiple α‐integrins by all‐atom replica exchange discrete molecular dynamics, competitive binding assays, and mutational analysis. We also demonstrated that CIB1 can bind to other whole integrin complexes in vivo using co‐immunopreceipitation assays. Isothermal titration calorimetry measurements indicated that this binding is driven by hydrophobic interactions and depends on residues in the CIB1 consensus binding site. These new mechanistic details of CIB1‐integrin binding imply that CIB1 could bind to all integrin complexes and act as a broad regulator of integrin function. Moreover, this work provides new insight into the nature of the protein‐protein interactions between CIB1 and its binding partners.

Grant Funding Source: Supported byNHLBI 5R01HL092544 and NIGMS 5K12GM000678
Ubiquitin‐like protein modification and proteasomes in Archaea (98.1)
Authors: Maupin‐Furlow Julie , Cao Shiyun , Chavarria Nikita , Fu Xian , Hepowit Nathaniel , Hwang Sungmin , Martin Jonathan , McMillian Lana , Miranda Hugo , Wu Yifei
Published: June 2021

Archaea encode a streamlined version of the eukaryotic ubiquitin‐proteasome system. In archaea, 20S proteasomes associate with AAA+ ATPases of simple subunit composition in the energy‐dependent degradation of proteins. More recently, we demonstrate that ubiquitin‐fold proteins of archaea (named small archaeal modifier proteins or SAMPs) are linked by isopeptide bonds to protein targets. This archaeal mechanism of attachment (named sampylation) appears less complex than ubiquitylation with a single ubiquitin‐activating E1 homolog (UbaA) functioning in the absence of ubiquitin‐conjugating E2 or ubiquitin‐ligase E3 enzymes. An archaeal isopeptidase composed of a single JAMM (JAB1/MPN/Mov34 metalloenzyme) domain is found to remove SAMPs from diverse protein conjugates, providing evidence that sampylation is reversible and likely regulated. Sites of sampylation, mapped to 36 lysines of 28 proteins by high‐throughput MS/MS analysis, were modeled in relationship to 3D‐structure and used to guide in vivo analysis and provide a basis for elucidating the biological function of sampylation. Based on this work, we propose that sampylation targets proteins for proteasome‐mediated proteolysis, modifies the catalytic active site of enzymes, modulates DNA‐binding protein interactions and regulates protein‐protein interactions.
Systems‐level analysis of xylose fermentation by experimentally‐evolved Saccharomyces cerevisiae (981.4)
Authors: Sato Trey , Parreiras Lucas , Ong Irene , Avanasi Ragothaman , Zhang Yaoping , Higbee Alan , Keating David , Coon Joshua , Gasch Audrey , Landick Robert
Published: June 2021

Experimental directed evolution and systems‐level analysis are powerful tools that can provide insights into the genetic regulation of biological and industrial processes. The fermentation of renewable plant biomass into cost‐effective biofuels is one such applied process. For the yeast biocatalyst, Saccharomyces cerevisiae, a major barrier to cellulosic ethanol production is its inability to rapidly ferment xylose from hemicellulose. To identify genetic factors regulating xylose fermentation, we performed directed evolution of an engineered S. cerevisiae strain on xylose and generated two evolved strains; GLBRCY127 was isolated by aerobic selection and a second was isolated from subsequent evolution of GLBRCY127 anaerobically. Metabolomic analyses between evolved and parent strains during the consumption of xylose from lab and cellulosic media suggested enhanced flux through the Pentose Phosphate Pathway and altered levels of TCA cycle intermediates. Genome sequencing and RNAseq analysis identified mutations in three genes that regulate mitochondrial function and signal transduction pathways. Importantly, deletion of these genes conferred xylose fermentation by the unevolved parental strain. This work identifies unexpected metabolic and physiological processes that limit cellulosic ethanol production and, when genetically modified, may help to realize a viable biofuels industry.

Grant Funding Source: Supported by DOE BER Office of Science DE‐FC02‐07ER64494.
Quantitative lc‐ms/ms analysis of cytoplasmic bacterial cell wall biosynthesis intermediates: application to vancomycin resistance VRE (982.2)
Authors: Vemula Harika , Putty Sandeep , Bobba Sudheer , Gutheil William
Published: June 2021

Vancomycin resistance in vancomycin‐resistant enterococci (VRE) is due to an alternative cell wall biosynthesis pathway in which D‐Ala‐D‐Ala is replaced, most commonly by D‐Ala‐D‐Lac. Methods for quantitative analysis of these intermediates has been lacking, which has impeded progress in characterizing the details of this resistance, and in developing new agent to counter this resistance.

Methods: LC‐MS/MS assays for both the alanine branch and the UDP‐linked cytoplasmic peptidoglycan intermediates have been developed. Alanine branch intermediates used Marfey’s reagent derivatization to enable analysis, and analysis of the UDP‐linked intermediates used an ion‐pairing approach. For the development of the UDP‐linked intermediate assays, these intermediate were purified from VRE using preparative ion‐ pairing HPLC. To demonstrate this assay, VRE was grown to log phase and treated with increasing concentrations of vancomycin.

Results: Analysis of alanine branch and UDP‐linked intermediates demonstrates the utility of LC‐MS/MS‐based methods for the study of the of the metabolite level changes in VRE in response to vancomycin.
Altered RAPD polymorphisms in Cordyceps sinensis analyzed with amplicon density‐weighted algorithms (988.1)
Authors: Zhu Jia‐Shi , Ni Luqun , Yao Yisang , Gao Ling , Li Yuling , Ma Shaoli , Wu Zimei , Tan Ningzhi , Wu Jian‐Yong
Published: June 2021

Objective: To examine the maturational alterations of RAPD polymorphism resulted from differential expressions of multi‐fungi in Cordyceps sinensis (Cs). Methods: Used 20 random primers and density‐weighted algorithms to analyze the integral polymorphic changes in Cs compartments during maturation; compared RAPD polymorphisms of Cs and Hirsutella sinensis (Hs). Results: Density‐unweighted algorithms neglected the differences in density of the DNA amplicons, while the density‐weighted algorithms integrated components of the amplicon density differences in similarity computations and cluster constructions. An overall similarity 0.42 was observed for Cs compartments at 3 maturation stages. The similarities for Cs stroma or caterpillar body at 3 maturational stages were 0.57 and 0.50, respectively. Cs compartments at 3 maturation stages formed a cluster. The low polymorphic similarities between Hs and Cs (0.55‐0.69) placed the Hs clade far from the Cs cluster, separated by the out‐group control Paecilomyces militaris. Conclusion: The wealthy RAPD polymorphisms are altering during maturationin the Cs compartments. The huge differences between the polymorphisms of Hs and Cs support the multiple fungi‐integrated micro‐ecosystem hypothesis for Cs, but do not support the hypotheses of “single fungal species” for Cs and the anamorph‐teleomorph connection between Hs and Cs.
Insulin signaling and copper homeostasis are functionally linked in 3T3‐L1 adipocytes (992.2)
Authors: Yang Haojun , Dhawan Neha , Ivy Kristin , Kaplan Jack , Lutsenko Svetlana
Published: June 2021

Copper is an essential element for human growth and development. Abnormal copper metabolism is associated with severe and potentially lethal disorders. Previous studies of copper misbalance in the animal model of Wilson’s disease have shown that copper levels influence cholesterol and triglyceride content in tissues. To understand the mechanisms connecting copper and lipid metabolism, we characterized copper homeostasis in 3T3‐L1 adipocytes upon differentiation and insulin signaling. We show that during adipogenesis copper uptake is upregulated 2‐3 fold. Protein levels of the copper‐transporting ATP7A also increase, presumably to facilitate copper delivery to semicarbazide‐sensitive amine oxidase (SSAO), a copper‐containing enzyme that exerts an insulin‐like effect and is highly upregulated in differentiated adipocytes. Both copper depletion and copper elevation inhibit SSAO’s activity, suggesting a fine‐tuned regulation of this enzyme by the metal. In turn, SSAO’s substrates and product regulate ATP7A localization in cells triggering trafficking form the trans‐Golgi network to vesicles. In response to insulin, copper uptake remains unchanged, whereas ATP7A traffics to the plasma membrane and vesicles indicating insulin‐dependent redistribution of intracellular copper pools. Altogether, our data revealed a novel metabolic connection between copper homeostasis and insulin signaling in differentiated adipocytes.

* Equal contribution

Grant Funding Source: NIH R01 GM101502
The effect of toxic heavy metals, Hg and Cd on the mRNA expression levels of the novel gene CIA7 of Chlamydomonas reinhardtii (995.4)
Authors: Sanchez Alvaro , Ynalvez Ruby
Published: June 2021

Heavy metal pollution of the environment is a growing concern to biological researchers due to its potentially harmful effects. Bioremediation of these toxic metals by species of algae has been researched as a possible alternative. Chlamydomonas reinhardtii contain a novel gene: CIA7of unclear function. Due to its possession of conserved cysteine amino‐acid residues, this gene is hypothesized to play a role in the binding and tolerance of heavy metals.

This study aims to qualitatively assess the levels of mRNA expression obtained from two strains of C. reinhardtii upon exposure to sub‐lethal levels of Hg and Cd. The two strains referred to are a wild‐type (D66) and a mutant strain (cia7) of C. reinhardtii. The mutant was isolated from inorganic carbon acquisition mutants. Upregulation of the CIA7 gene following a toxic metal treatment will provide a basis for CIA7’s role in heavy metal tolerance.

To perform the expression analysis of the CIA7 gene, D66 and cia7 algal strains were exposed to sub‐lethal concentrations of Hg and Cd in liquid media. A separate control treatment with no heavy metal content was also performed. After an exposure time of three hours, total RNA was extracted and purified. The entire RNA content of the algae was then converted into its complementary DNA (cDNA). Amplification of the CIA7 gene was performed using the polymerase chain reaction and the band intensities were compared using Quantity One analysis software. The comparison in expression levels between both algal strains and between metal treatments will help determine whether CIA7 has the potential for abatement of toxic heavy metals.

Grant Funding Source: Supported by: Norman Hackerman Advanced Research Program
Mcm10 Interacts with Mrc1 and May Play a Possible Role in DNA Damage Checkpoint Pathway (LB123)
Authors: Hendrix Chance , Fultz Brandy , Das‐Bradoo Sapna
Published: June 2021

To ensure genome integrity, cells must replicate with absolute fidelity. However, cells are constantly under stress that causes DNA damage or block replication. To circumvent these problems, cells have a quality control system termed DNA damage checkpoint. In humans, defects in this checkpoint cause genome instability and a strong predisposition to cancer. A recent study has identified minichromosome maintenance protein 10 (Mcm10) as one of the few replication proteins that prevent DNA damage. However, whether Mcm10 has an active role in preventing DNA damage is unknown. We have observed that Mcm10 and mediator of replication checkpoint 1 (Mrc1) interact in S. cerevisiae whole cell extracts. Moreover, a robust interaction was observed between Mcm10 and Mrc1 by yeast two‐hybrid assays. Our data suggests Mcm10 interacts through the conserved C‐terminus of Mrc1. Interestingly, it is the N‐terminus of Mcm10 that binds to Mrc1. We are currently conducting experiments to further elucidate this interaction. These studies promise to identify a novel role of Mcm10 in DNA damage response pathway and maintenance of genomic integrity.

Grant Funding Source: Supported by the National Institute of General Medical Sciences of the National Institutes of Health through Grant Number 8P20GM103447 to SDB.
Potential association between irritable bowel syndrome and the gene encoding human intestinal sucrase‐isomaltase (LB131)
Authors: Diekmann Lena , Kuech Eva‐Maria , Phillip Ute , Distl Ottmar , Money Mary E. , Naim Hassan Y.
Published: June 2021

Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder characterized by abdominal pain and alterations in bowel pattern with a prevalence of 10‐15 % in humans. The molecular basis of IBS is not well understood.

We sequenced the coding region of the sucrase‐isomaltase gene (SI) in a mother and her two children who suffer from IBS. The only polymorphism we found to be homozygous or heterozygous was Val15Phe (V15F, rs9290264, g.7526G>T, c.43G>T). We assessed the role of this exchange at the biochemical, cellular and functional levels by expressing SI containing V15F (SI‐V15F) in COS‐1 cells.

The biosynthesis, glycosylation and folding of SI‐V15F were essentially comparable to those in wild type SI. The substitution of Val in the first α‐helix of the transmembrane domain of SI by a bulky Phe may have implications on the mode of association of SI with the inner leaflet of the membrane. For this, we investigated the association of SI‐V15F with lipid rafts by assessment of its solubility towards the non‐ionic detergent Triton X‐100 (TX‐100). In comparison to wild type SI, SI‐V15F is more soluble in TX‐100, i.e. less associated with lipid rafts. Cell surface biotinylation indicated that the trafficking of SI‐V15F to the plasma membrane is reduced by almost 20 % as compared to wild type SI. Finally, SI‐V15F revealed a 35 % decreased sucrase activity.

Altogether, the combinatorial effects of reduced cell surface expression and enzymatic activity levels lead to a substantial decrease in the overall digestion capacity of SI‐V15F and strongly suggest an association between IBS and the substitution V15F.
Small transmembrane protein inhibitors of the platelet‐derived growth factor β receptor (LB215)
Authors: Petti Lisa , Talbert‐Slagle Kristina , Chacon Kelly , Hochstrasser Megan , DiMaio Daniel
Published: June 2021

The platelet‐derived growth factor β receptor (PDGFβR) tyrosine kinase has been implicated in several cancers and is normally activated by binding of ligand to its extracellular domain. In addition, the small E5 transmembrane protein of bovine papillomavirus transforms cells by binding to the transmembrane domain of the PDGFβR and inducing receptor dimerization and activation. By screening retroviral libraries expressing hundreds of thousands of small proteins with randomized transmembrane domains, we identified artificial small transmembrane proteins termed “traptamers”, which specifically activate the PDGFβR and transform cells even though they have little or no sequence similarity to E5. A single conservative amino acid substitution in two traptamers converts them into inhibitors of the PDGFβR. The mutant traptamers bind to the PDGFβR and inhibit the mitogenic and transforming effects of PDGF and its homologue v‐sis by preventing phosphorylation of the receptor at particular tyrosine residues, suggesting that they may alter the conformation of the PDGFβR and/or its interaction with certain signaling proteins. These results validate a new strategy for isolating highly specific short transmembrane protein inhibitors of growth factor receptors, which could be used to gain new insight into growth factor receptor signaling and possibly as prototypes for the development of novel therapeutics.

Grant Funding Source: Supported by a grant from the National Cancer Institute (CA37157)
Two new anchoring factors, GSKIP and GSK3beta join to strengthen cAMP/PKA/Drp1 axis signaling under oxidative stress (LB221)
Authors: Hong Yi‐Ren
Published: June 2021

Two new anchoring factors, GSKIP and GSK3beta join to strengthen cAMP/PKA/Drp1 axis signaling under oxidative stress Yi‐Ren Hong1 1Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan We firstly demonstrated that PKA, GSKIP, GSK3β and Drp1 form a working complex which similar to proclaimed cAMP/PKA/Drp1 axis that has been linked to elongated mitochondrial morphology (JBC, 2007; Nat Cell Biol, 2011). This finding raises a question about whether GSKIP and GSK3β as mediators of cAMP/PKA/Drp1 axis signaling. Our data demonstrated that overexpressed GSKIP and its mutants, Drp1 Ser637 enhanced phosphorylation 7~8‐fold in the GSKIP wt group compared to both defective mutant groups in a temporal manner under H2O2 and forskolin challenge, implicating both V41/L45 (PKA RII binding) and L130 (GSK3β binding) are important to the Drp1‐associated protective action of GSKIP. In addition, our data also imply that GSK3β may act as a scaffold protein that recruits Drp1 for PKA, directly phosphorylating at Ser637. We therefore silenced either of GSKIP or of GSK3β and resulted in a dramatic decrease of Drp1 Ser637 phosphorylation, indicating that both GSKIP‐ and GSK3 plays as anchoring protein in this novel PKA/GSKIP/GSK3/Drp1 complex. By using confocal fluorescent microscopy, only GSKIP wt, but not V41/L45 or L130 mutant, exhibited elongated mitochondrial morphology after compromising the effects of H2O2 and forskolin. Taken together, in addition to involvement in neurite outgrowth, our data illustrated a new role for GSKIP, associated with acquired resistance to H2O2‐induced apoptosis through influencing mitochondrial morphology by coordinating both the activation of PKA‐mediated Drp1 phosphorylation at Ser637 and tethering GSK3β to recruit Drp1 by anchoring rather than by kinase activity.
Autophosphorylation by the Histidine kinase CheA (LB222)
Authors: Greenswag Anna , Crane Brian
Published: June 2021

Chemotaxis is the signal transduction mechanism that allows bacteria to change direction in response to an external stimulus. Detection of an environmental stimulus by the receptors has a cascading effect leading to the flagellar motor changing direction. This results in the tumbling of the bacterium. The receptors form hexagonal lattices at the poles of cells. The histidine kinase CheA and coupling protein CheW are localized to the lattice to form the ternary complex. CheA, the principal enzyme in the chemotaxis signaling pathway, is a multi domain protein comprised of: P1‐histidine phosphotransfer, P2‐regulatory, P3‐dimerization, P4‐kinase, and P5‐regulatory. From previous studies it is known that the transfer of the γ‐phosphate of ATP occurs between the P1 and P4 domain, however how autophosphorylation activity is regulated is not completely understood.

To comprehend this transient interaction, variants (ΔP2) of Thermotoga maritima CheA were generated. Small angle X‐ray scattering (SAXS) data has shown that spatial changes occur upon addition of an ATP analog. To determine relative activity of the ΔP2 variants or CheA domains radioisotope assays were used. Point mutations on the full length and ΔP2 CheA showed whether autophosphorylation occurs cis or trans in the dimer. Crosslinking between residues near the active sites in the P4 and P1 domains enabled isolation of the autophosphorylation transfer conformation. The crystal structure of P3P4 domains reveals a more active configuration of the P4 domain. Progress towards understanding how the domains interact to facilitate the transfer of the ATP γ‐phosphate is not clear and will be discussed.
Molecular mechanism underlying nuclear localization of EBP50 (LB225)
Authors: Lim Hooi‐Cheng , Jou Tzuu‐Shuh
Published: June 2021

Ezrin‐radixin‐moesin (ERM)‐binding phosphoprotein of 50 kDa (EBP50) is a protein that is expressed at the apical surface of luminal organs. EBP50 consists of two tandem PDZ domains and a C‐terminal ezrin biding site, and this architecture allows EBP50 to organize molecules into functional complexes at the membrane periphery. Therefore, the studies of EBP50 were once focused on its regulatory roles at the plasma membrane, such as ion transporters activation, receptors recycling, and microvillar assembly. Later, EBP50 was found localized at the nucleus of cultured epithelial cells and carcinoma specimens, and interacted physically with nuclear proteins. However, the molecular mechanism that regulates nuclear localization of EBP50 remains unclear. A RNA interference‐based screening that coupled with image analysis was carried out to discover signaling pathways specific for trafficking of EBP50. The downstream effector of the mitogen‐activated protein kinase (MAPK) signaling pathway, p90 ribosomal S6 kinase alpha 1 (RSK1) was identified as a potential regulator, in which knockdown of RSK1 reduced the nuclear accumulation of EBP50. Furthermore, in vitro pull‐down assay and co‐immunoprecipitation approach had shown that RSK1 binds to the first PDZ domain of EBP50. RSK phosphorylates its substrate at RXRXXpS/T consensus phosphorylation motif. In vitro kinase assay showed that epidermal growth factor‐activated RSK1 phosphorylates EBP50, at the RXRXXpS/T motif. Collectively, our study identified RSK1 as a novel interacting partner of EBP50 that phosphorylates EBP50 at the RXRXXpS/T motif.

Grant Funding Source: Supported by Taiwan National Science Council
Conformational switching in a copper‐sensing operon repressor (CsoR) (LB246)
Authors: Chang Feng‐Ming , Coyne H. , Fang Xianyang , Wang Yun‐Xing , Dann Charles , Giedroc David
Published: June 2021

Copper is an essential transition metal for living organisms but it is detrimental in excess. The metalloregulatory protein copper‐sensing operon repressor (CsoR) in bacteria has evolved to prevent cytoplasmic copper toxicity. Cu(I)‐binding to tetrameric CsoRs mediates transcriptional derepression of copper resistance genes but the mechanism is unknown. A phylogenetic analysis of 227 DUF156 protein members including biochemically or structurally characterized CsoR/RcnR repressors reveals that Geobacillus thermodenitrificans (Gt) CsoR characterized here is representative of CsoRs from pathogenic bacilli Listeria monocytogenes and Bacillus anthracis. The 2.56 Å structure of Cu(I)‐bound Gt CsoR reveals that Cu(I) binding induces a kink in the α2‐helix between two conserved copper‐ligating residues and folds an N‐terminal tail (residues 12‐19) over the Cu(I) binding site. NMR studies of Gt CsoR reveal that this tail is flexible in the apo‐state with these dynamics quenched upon Cu(I) binding. Small angle X‐ray scattering (SAXS) experiments on an N‐terminally truncated Gt CsoR (∆2‐10) reveal that the Cu(I)‐bound tetramer is hydrodynamically more compact than is the apo‐state. A mutational analysis of residues critical to N‐terminal tail folding reveals that these residues function to stabilize the apoprotein‐DNA complex and/or control the extent of allosteric negative regulation of DNA binding by Cu(I), but to varying degrees. The mechanism of Cu(I)‐mediated allosteric switching in CsoRs is discussed.

Grant Funding Source: Supported by NIH grant GM042569
Design of a Stimulus‐responsive Human Serum Albumin‐based Nanoparticle for Photodynamic Therapy (LB249)
Authors: Molina Anna , Benítez Marimar , Berríos Kiara , Griebenow Kai
Published: June 2021

Photodynamic therapy (PDT) is a promising treatment for cancer. It relies on the accumulation of a photosensitizer (PS) drug in the target area and the subsequent irradiation with a light source to induce the formation of reactive oxygen species that cause cell death. The incorporation of these drugs in drug delivery systems (DDS) has proven to increase selectivity and overall efficiency of the therapy in vivo. We have synthesized a new DDS using human serum albumin nanoparticles (HSA NPs) of a discrete size as carriers. The PS drug Chlorin e6, which has proven to be effective for PDT, was covalently linked onto the NPs to prevent non‐selective release. Our NPs were stabilized with a redox‐sensitive crosslinker for targeted activation of the drug in a reducing environment, such as that of the cell cytoplasm. Absorbance and fluorescence spectroscopy experiments showed that the NPs disintegrated and that the PS photoquenching decreased in a 10 mM glutathione solution, resulting in the activation of the drug. The system was physically characterized in terms of size, shape and drug loading. The size obtained (280‐300 nm) was appropriate for accumulation in cancer tissue. Cell internalization and phototoxicity of the system were tested in HeLa cells by cell viability assays and confocal microscopy, showing efficient accumulation of the drug and dose‐dependent cytotoxicity. Our system demonstrated to be redox‐activated, with efficiency comparable to the free drug in vitro.

Grant Funding Source: Supported by NIH Research Initiative for Scientific Enhancement (RISE) Program with grant number: 2R25GM061151‐12.
Interaction of Fasciola hepatica Fatty Acid Binding Protein with TLR‐2: A preliminary study to understand the immunomodulation mechanisms that exert F. hepatica on the immune system (LB287)
Authors: Ruiz‐Jiménez Caleb , Espino Ana
Published: June 2021

In the last decade scientists have shown interest to the strong anti‐inflammatory role that exhibit some helminthes parasites. Fasciola hepatica, is able to induce long‐lasting chronic disease in the mammalian host associated to predominant Th2 responses, which is linked to an anti‐inflammatory phenotype. For this purpose, parasite secrete and express on its surface a large number of proteins that are necessary for their metabolism, and, in addition, it has the ability to modulate the host’s immune response. One of these proteins is fatty acid binding protein (FABP), a protein family of 12kDa that play an essential role in the lipid metabolisms. Recently, our group demonstrated that FABP is able to block the inflammatory cytokine storm typically induced by LPS from Gram‐negative bacteria through the Toll‐like receptor 4 (TLR‐4) and thereby is able to prevent the septic shock in mice. In this work, using HEK‐TLR2 and THP1‐Blue CD14 cells we demonstrated that FABP is also able to block the activation of TLR2 typically produced by specific TLR2‐ligand in a manner that is dose dependent. In addition, in all cases where cells were exposed to FABP prior stimulation with specific TLR2‐ligand the production of inflammatory and pro‐inflammatory cytokines resulted in significant suppression. These results demonstrate that FABP may have a suppressive effect on cells of immune system targeting multiple TLR pathways.
Geometric factors influencing hydrogen‐deuterium exchange: Detection of mobile water molecules in the peptide‐MHCI ‐ binding interface (LB291)
Authors: Jin Yining , Patil Sachin , von Grafenstein Hermann
Published: June 2021

Hydrogen deuterium exchange (HDX) has long been used as an important technique to study conformational changes of proteins in solution, protein folding as well as protein ligand interactions. HDX is thought to detect exposure of protein backbone amide (bNH) hydrogens to solvent or internal water. However, it is not known how this exposure is defined in geometric terms, especially for internal water‐filled cavities. The experiments described here was designed to gain a better understanding of the influence of factors like distance and angle of orientation of bNH‐groups relative to internal water pockets. We used Major Histocompatibility Class I (MHCI)‐peptide complexes as a model to address this question. We expressed MHCI heavy and light chains and reconstituted MHCI‐peptide complexes with synthetic immunogenic peptides. We measured HDX using LC‐MS, and characterized structural parameters of water‐filled pockets with the help of computer programs like Pymol and Surfnet. We have successfully developed a model combining both distance and orientation of bNHs relative to nearby gaps which can predict HDX results and explain the HDX data achieved from the LC‐MS experiments. In addition to identifying geometric factors determining HDX, the project is also the first time attempting to correlate peptide‐MHC interface gap properties to the experimental H/D‐exchange in a quantitative manner. These results can be further applied to any other receptor ligand binding system which involves water molecules.

Grant Funding Source: Supported by NIH and University of Toledo Financial Award
Antibacterial activities and chemical characteristics of usnic acid and other secondary metabolites extracted from Flavoparmelia caperata lichen species using methanol as a solvent (LB293)
Authors: Bixho Ida , Scott Elizabeth , Berger Michael
Published: June 2021

It is known that usnic acid extracted from Icelandic lichens has antimicrobial properties (Ingolfsdottir, 2002). The goal of this project is to investigate the antibacterial activities of the metabolites of the lichen Flavoparmelia caperata found growing in New England. Chemical extractions of lichen metabolites were performed using methanol as a solvent. Following the extraction of the lichen metabolites, their antimicrobial properties were tested using in vitro antimicrobial assays against the Gram positive Staph aureus, and Gram negative E. coli and Ps. aeruginosa. In addition, the antibacterial activities and chemical characteristics of lichens growing in shade were compared to those growing in the sun, based on literature finding that suggest that an increase in usnic acid is influenced by various ecological factors such as light and temperature (Cochietto, 2002). Following extraction, the secondary metabolites of lichens were identified using GC‐MS. Early results indicate that shade‐grown lichens contain larger quantities of usnic acid, and sun‐grown lichens contain smaller quantities of usnic acid in addition to other acids. Moreover, there are some indications that Staph aureus is more sensitive to the sun‐grown lichen. This may be due to the synergic effects of usnic acid and friedelan‐3‐one acid found in the sun‐grown lichen.
Differential regulation of adipokine levels after Bariatric surgery (LB345)
Authors: Wijayatunga Nadeeja , Sam Valerie , Blackledge Camille , Siriwardhana Nalin , Mancini Matthew , Mancini Gregory , LeMieux Monique , Moustaid‐Moussa Naima
Published: June 2021

Bariatric surgery is known to cause significant weight loss, reduction in insulin resistance, cardiovascular disease and mortality. We hypothesized that adipose and systemic inflammation will be decreased post bariatric surgery. Our objective was to study changes in adipokine levels following bariatric surgery. We recruited patients undergoing Roux En Y gastric bypass and laparoscopic gastric band placement. Demographic data and both serum and adipose tissue samples were collected at the time of surgery, 2 weeks and 6 months post operatively. Luminex Human cytokine and adipokine kits were used along with adiponectin, Tumor Necrosis Factor‐α (TNF‐α), leptin and Monocyte Chemotractant Protein‐1 (MCP‐1) ELISAs to measure levels of these cytokines in serum and/or adipose tissues. Percentage changes in adipokines were calculated from time of surgery to each time point after surgery. There was a significant increase in adipose adiponectin at each post‐surgery time. Serum adiponectin showed an increasing trend by 6 months, but was not significant. Serum leptin, TNFα and MCP‐1 all showed decreasing trends from time of surgery to 6 months post surgery, but differences were not statistically significant. The percentage reduction for MCP‐1 at 6 months was significantly higher than for TNF‐α. These findings suggest that serum and adipose adipokine levels are differentially regulated by bariatric surgery. Additional studies are necessary to determine how these changes in tissue and circulating cytokine levels relate to various bariatric surgery outcomes.

Grant Funding Source: PMERF (Physician’s Medical Education and Research Foundation, Knoxville, TN) and VPR and COHS Texas Tech University startup funds, Lubbock, TX
Osteological change in the auricular surface: a comparison of two aging methods (LB37)
Authors: Godde Kanya , Hens Samantha
Published: June 2021

Senescence changes in the auricular surface are best described by the two vastly different techniques, Lovejoy et al. (1985) and Buckberry and Chamberlain (2002). While the Lovejoy et al. method combines changes in various traits into a linear progression of phases, the Buckberry and Chamberlain approach scores changes in individual traits separately to create a composite score. Here, we attempt to test for differences in accuracy between the two methods using a Bayesian approach. The Buckberry and Chamberlain method was created using this statistical modeling, and thus, we hypothesize it will outperform Lovejoy et al.’s technique. Three modern populations of males were utilized to model aging for both methods: Americans (reference sample), Thai (informative prior), and Portuguese (target sample). A Bayesian transition analysis was applied to estimate the age‐of‐transition for each phase in each technique. Cumulative binomial tests assessed the accuracy of age ranges. Overall, both methods performed similarly under statistical scrutiny, but the Lovejoy et al. method performed significantly lower than probability without the application of a statistical age estimation method. Thus, although applying transition analysis with a Bayesian component will provide the best age estimates, the Buckberry and Chamberlain method can be applied accurately without additional statistical analysis.
The Effect of Gender and National School Lunch Program Participation on Saturated Fat Content of Entrée Items Chosen by Elementary School‐Aged Students (LB379)
Authors: Anderson Annie , Granberg Ellen , Kropp Jaclyn , Peckham Janet , Condrasky Marge , Mroz Tom , Haley‐Zitlin Vivian
Published: June 2021

□ The purpose of this study was to examine participants in a National School Lunch Program (NSLP) school district, and the effect of gender or participation in the NSLP on the selection of high saturated fat content entrée items by the elementary school students. Nutrition information provided to parents often does not include a profile of the fats offered in school lunch items. This was true of this study’s schools. However, new NSLP guidelines focus upon saturated and trans fat content in school lunches. The total kcal limit is <30%/total kcal, and saturated fat is <10%/total kcal, along with 0gms/serving trans fat. So, there is a need to monitor the fat profile of foods offered to ensure schools meet the current guidelines. As a part of a large plate waste study, all entrée items (79, 360) served in February 2013 in 11 elementary schools were analyzed for SFAs, MUFAs, PUFAs, trans fat, total fat and total kcal using “best fit” selection criteria with Nutritionist Pro TM nutritional analysis software based upon matching of nutrient content (kcal, CHO, protein, total fat, and sodium) from the school district and recipes of actual entrée items served. In the offer‐based school lunch service provided, three entrée items/day were served with a constant of vegetarian choice (consisting of cheese stick, yogurt, and cracker product) each day. Student numbers were 5,375 total; and a total of 41, 739 selections were made by males and 37, 621 by females. Total “paid”, “free” and “reduced” meals were 24,654; 50,366; and 4,340, respectively. Percentage of “free”, “reduced” and “paid” status meals for males were 63%, 5%, and 32% respectively and for females were 64%, 5.5%, and 30.5%. Point of Sale data collected during the same time period coupled with lunch dietary analysis data is being used to determine the effect of gender and/or participation in the free and reduced school lunch program on the selection of high saturated fat content entrée items by elementary school aged students and will be presented.

□

Grant Funding Source: CBBS Health Initiative Pilot Grant, Clemson University
Pharmacokinetics and plasma cellular anti‐oxidative effect of flavanols after oral intake formulating green tea with vitamin C and xylitol in healthy subjects (LB387)
Authors: Shim Soon‐Mi
Published: June 2021

Soon‐Mi Shim Department of Food Science & Technology and Carbohydrate Bioproduct Research Center, Sejong University, 98 Gunja‐dong, Gwangjin‐gu, Seoul, 143‐747, Republic of Korea The objective of the current study was to characterize pharmacokinetic, plasma cellular antioxidant activity and plasma metabolites of flavanols after oral intake of green tea formulating with vitamin C and xylitol in healthy subjects. Twelve healthy subjects took three green tea tablet (GT) with 100 mL of water and crossed‐over to intake three green tea tablet formulating vitamin C and xylitol (GTVX) with 100 mL of water following a 2‐week wash out period. Liquid Chromatography‐Mass Spectrometry (LC‐MS) was carried out for analysis flavanols in plasma taken at certain time points. The plasma flavanols’ concentration‐time profile for each study subject was determined by using a noncompartmental approach. Total Radical‐Trapping Antioxidant Parameter (TRAP) method was used for measuring total antioxidant activity (TAC) of plasma. Cmax, Tmax, AUC, and t2/1 of favanols in plasma after consumption of GTVX were 2.14 h, 5980.58 ng/mL, 18915.56 h*ng/mL, and 1.13 h, respectively, indicating that GTVX showed higher AUC than GT. The peak of trolox value was found at 3 and 0.5 hr after intake of GT and GTVX, respectively, revealing that they were different from Tmax of total flavanols. TAC of plasma was found to be significantly higher in GTVX than GT at each time point. Measured TAC of plasma was highly correlated with metabolites to gallated flavanols rather than total level of flavanols. Results from our study suggest that formulating green tea with vitamin C and xylitol could increase bioavailability of flavanols, resulting in enhanced biological function such as cellular anti‐oxidative effect.

Grant Funding Source: Supported by Korea government (MEST)
Effect of Transforming Growth Factor‐β (TGF‐β) on Sox9 function and chondrocyte phenotype (LB40)
Authors: Perez Jessica , Seo HwaSeon , Serra Rosa
Published: June 2021

TGF‐β enhances Sox9 protein expression as well as downstream targets that regulate extracellular matrix production in bovine articular chondrocytes (BACs). The extracellular matrix protein 3‐Prime‐Phoshoadenosine 5‐Prime‐Phosphosulfate Synthase 2 (Papss2) has been associated with spondyloepimetaphyseal dysplasias in humans and is required for proper sulfation of proteoglycans in cartilage. Our work and that of others has shown that TGF‐β can regulate Papss2 expression in articular cartilage. We aim to identify whether Sox9 is involved downstream of TGF‐β in the regulation of Papss2 mRNA expression.

Using primary BACs in micromass cultures, we analyzed the effect of TGF‐β (5ng/ml) on chondrocyte phenotype, Sox9 protein expression, stability, cellular localization and activity. We also assessed the effect of TGF‐β and/or Sox9 overexpression on the mRNA expression of Papss2, Prg4 and GAPDH via quantitative real‐time PCR.

We found that treatment with TGF‐β enhanced proteoglycan content and delayed chondrocyte hypertrophic differentiation in BACs. We also observed an increase in Sox9 protein levels and stability, without changes in mRNA. The increase in Sox9 protein correlated with enhanced Col2a‐reporter activity. Importantly, we found Papss2 to be upregulated via Sox9 when wild‐type (WT)‐Sox9 was overexpressed via adenoviral transduction (50 MOI/cell). Interestingly, treatment +/‐ TGF‐β when Sox9 was overexpressed did not further increase Papss2 mRNA, supporting the hypothesis that Sox9 works downstream of TGF‐ β. However, TGF‐β, but not Sox9, increased Prg4 mRNA expression, suggesting specificity of signaling. A GAPDH control was examined and remained unchanged in response to either treatment. The regulation of Papss2 by Sox9 represents a novel paradigm involving TGF‐β‐dependent signaling in articular chondrocytes.

Grant Funding Source: Supported by NIAMS‐ NIH