bluuebunny/crossref_metadata_2025 huggingface dataset:
Relative quantification of the effect of blood meal iron on protein expression in the ovaries of Aedes aegypti (778.8)
Authors: Geiser Dawn , Li Wenzhou , Breci Linda , Winzerling Joy
Published: June 2021

Diseases transmitted by mosquitoes are estimated to kill more than 2 million people annually. The vector for dengue, Aedes aegypti (yellow fever mosquito), is found in the desert Southwest of the continental United States. One approach to limiting the spread of disease is to reduce the numbers of mosquito vectors. Our area of research is iron metabolism. Our long term goal is to use molecular techniques to alter the expression of iron related proteins in mosquitoes to reduce mosquito fecundity and survival. Our hypothesis is that female mosquitoes require iron in the blood meal as an essential nutrient to aid in egg development. Although the iron is necessary for fecundity it is also highly oxidative and toxic in biological systems unless the mineral is strictly regulated to mitigate its oxidative potential. We are currently analyzing tissue‐specific, iron regulated proteins of the mosquito ovary 24 h post‐blood meal fed on artificial blood meal diets supplemented with no iron or low iron, or a porcine blood meal (high iron). Measurement of the blood meal iron effect on ovarian protein expression was carried out using Tandem Mass Tags (TMT) which permits simultaneous identification and relative quantification of proteins during LTQ Velos Orbitrap LC‐MS/MS analysis. This research will contribute to our understanding of mosquito development in general, as well as ovarian iron metabolism.

Grant Funding Source: Supported by funds from the Arizona Ag Experiment Station and CALS
Bioinformatic characterization of Lucina pectinata hemoglobins (778.9)
Authors: Montes‐Rodríguez Ingrid , Ropelewski Alexander , Nicholas Hugh , Cadilla‐Vazquez Carmen L. , González‐Méndez Ricardo , Lopez‐Garriga Juan
Published: June 2021

Lucina pectinata is a bivalve clam that is found in sulfide rich muds environments and lives in symbiosis with chemoautotrophic bacteria. Three different hemoglobins have been described in L. pectinata and in addition to their physiological role in the clam, are believed to be responsible for transportation hydrogen sulfide (H2S) and oxygen (O2) to a chemoautotrophic bacterial symbiont. Hemoglobin I (HbI) is a sulfide‐reactive hemeprotein while Hemoglobin II (HbII) and Hemoglobin III (HbIII) are oxygen‐reactive hemeproteins. Multiple sequence alignment analysis carried with L. pectinata hemoglobins and other mollusk globin sequences showed several conserved residues within this subfamily. Motif analysis using MEME also revealed a motif shared only between HbII and HbIII sequences. A phylogenetic tree was constructed the 19 molluscan globin sequences obtain after doing the CD‐Hit clustering. The tree separated them into three main clusters, a cluster for the class of Pteriomorphia, another for Heteroconchia and other for Gastropods. Hemoglobins of L. pectinata, which are found in the heteroconchia cluster, are group with the globins of those of the Calyptogena sp, where HbI is more related to this species while HbII and HbIII seen to be more distant. Based on the phylogenetic analysis and the reconciliation with the species tree, we suggest that a first gene duplication at an early evolutionary stage resulted in the sulphide binding and oxygen binding genes. A more recent gene duplication of the oxygen binding gene occurred to give rise to two oxygen binding proteins, HbII and HbIII. This hypothesis is supported with the fact that HbI shows low sequence homology to both HbII and HbIII, while there are more similarities between HbII and HbIII. In addition, HbI appears to have been changing over time, apparently not subject to the constraint of binding oxygen and acquired a unique function for a specialized environment.
Screening fluorescent voltage probes with spontaneously spiking HEK cells (779.5)
Authors: Park Jeehae , Werley Christopher , Cohen Adam
Published: June 2021

A major difficulty for imaging brain activity comes from the lack of voltage‐sensitive probes that have fast, sensitive and bright characteristics. Unlike other probe development challenges, screening fluorescent voltage probes has been hampered by the low throughput of patch‐clamp characterization. We introduce a line of non‐fluorescent HEK cells that stably express NaV 1.3 and KIR 2.1 and generate spontaneous electrical action potentials. These cells enable rapid, electrode‐free screening of speed and sensitivity of voltage sensitive dyes or fluorescent proteins on a standard fluorescence microscope. We screened a small library of mutants of Archaerhodopsin 3 (Arch) in spiking HEK cells and identified two mutants with greater voltage‐sensitivity than found in previously published Arch voltage indicators.
Identification of ectonucleotide pyrophosphatase/phosphodiesterase 3 as a new modifier of glycan biosynthesis (788.3)
Authors: Taniguchi Naoyuki , Korekane Hiroaki , Park Jong Yi , Matsumoto Akio , Nakajima Kazuki , Takamatsu Shinji , Ohtsubo Kazuaki , Miyamoto Yasuhide
Published: June 2021

Our group and Pierce’s group reported that a brain‐specific β1,6‐N‐acetylglucosaminyltransferase GnT‐IX (GnT‐Vb) has a broad transfer activity toward N‐linked and O‐mannosyl glycans. We showed that its gene expression is regulated by epigenetic histone modifications. We also found that GnT‐IX‐null mice showed enhanced remyelination with impaired astrocyte activation in the cuprizone‐induced demyelination model. Here we show the existence of an endogenous inhibitor for GnT‐IX in Neuro2a (N2a) cell. We purified this inhibitor from N2a cells and found that it is identical to ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) by mass spectrometry. The underlying mechanism of GnT‐IX inhibition was found to be the ENPP3‐catalyzed hydrolysis of the nucleotide‐sugar donor substrate, UDP‐GlcNAc. Indeed, the ENPP3‐knockdown cells exhibited significantly increased levels of intracellular nucleotide sugars that suggests a biological function of ENPP3 in the regulation of the intracellular nucleotide sugar levels. In addition to chaperones or other known regulators of glycosyltransferases, the ENPP3‐mediated hydrolysis of nucleotide sugars may have significant impacts on other glycosyltransferase activities responsible for altering the total cellular glycosylation profile and modulating cellular functions.
Congenital disorder of glycosylation caused by a mutation in SSR4, the signal sequence receptor 4 protein of the TRAP complex (789.3)
Authors: Losfeld Marie‐Estelle , Ng Bobby , Kircher Martin , Buckingham Kati , Turner Emily , Eroshkin Alexey , Smith Joshua , Shendure Jay , Nickerson Deborah , Bamshad Michael , Freeze Hudson
Published: June 2021

Congenital Disorders of Glycosylation (CDG) are a family of genetic disorders affecting the glycosylation pathway or the trafficking of glycoproteins. Nearly 50 genes causing CDG have been described, but many patients biochemically diagnosed with CDG do not have mutations in known genes. Among our CDG patients, we identified by exome saequencing, in three patients, new variants of the X‐linked SSR4 gene which encodes a protein of the heterotetrameric translocon‐associated protein (TRAP) complex. Two of these variants results in the absence of SSR4 expression. In these cases, we observed that expression of other TRAP complex proteins was also reduced. The glycosylation marker Glyc‐ER‐GFP was used to confirm the underglycosylation in fibroblasts from patients. Over‐expression of the wild type SSR4 allele partially restores glycosylation of the marker and expression of the other members of the TRAP complex. This is the first evidence that the TRAP complex, which binds to the oligosaccharyltransferase complex, is directly involved in N‐glycosylation.

Grant Funding Source: Supported by the National Institutes of Health grant [R01DK55615] and “The Rocket Fund”.
Genetics and virulence role of the classical group A Streptococcus Lancefield antigen (790.2)
Authors: Cole Jason , van Sorge Nina , Kuipers Kirsten , Henningham Anna , Aziz Ramy , Kasirer‐Friede Ana , Lin Leo , Berends Evelien , Davies Mark , Dougan Gordan , Zhang Fan , Dahesh Samira , Shaw Laura , Gin Jennifer , Cunningham Madeleine , Merriman Joseph , Rooijakkers Suzan , Malley Richard , Walker Mark , Shattil Sanford , Schlievert Patrick , Choudhury Biswa , Nizet Victor
Published: June 2021

All strains of group A Streptococcus (GAS), a leading cause of infection‐related mortality worldwide, express group A carbohydrate (GAC), comprising a polyrhamnose backbone with an immunodominant N‐acetylglucosamine (GlcNAc) side chain. The immunodominant GlcNAc epitope of GAC is the basis of all rapid diagnostic testing for GAS infection, but a function for this eponymous antigen in disease pathogenesis has never been proposed. Here, we identify the genetic basis of the GAC GlcNAc side chain and generate an isogenic mutant for analysis of its role in virulence and immunogenicity. Loss of the GlcNAc epitope on GAC attenuated GAS virulence in two animal infection models, in association with increased sensitivity to killing by whole blood, neutrophils, and platelet‐derived antimicrobials. Antibodies generated against the modified GAC structure promoted opsonophagocytic killing of multiple GAS serotypes and protected against systemic GAS challenge in mice after passive immunization. Our results demonstrate how functional insight into the classical Lancefield antigen has implications for GAS disease pathogenesis and vaccine development.

Grant Funding Source: National Institutes of Health; National Health and Medical Research Council of Australia
Disruption of the nuclear membrane by the antimicrobial peptide LL‐37 leads to the formation of neutrophil extracellular traps (793.1)
Authors: Neumann Ariane , Völlger Lena , Nerlich Andreas , Meerloo Timo , Gallo Richard , Nizet Victor , Naim Hassan , von Köckritz‐Blickwede Maren
Published: June 2021

Neutrophil extracellular traps (NETs) have been described as a fundamental innate immune defense mechanism. During formation of NETs the nuclear membrane of mammalian cells is disrupted by a yet unknown mechanism. The cathelicidin LL‐37 has been shown to bind and damage membranes deficient in cholesterol and sphingomyelin such as bacterial membranes. Consequently, we hypothesized that LL‐37 could potentially damage the nuclear membrane, which contains reduced amounts of cholesterol, and thereby facilitates the formation of NETs by activated neutrophils.

Different concentrations of LL‐37 were used to stimulate primary blood‐derived neutrophils. The percentage of NET‐releasing cells was calculated using immunofluorescence microscopy and revealed that LL‐37 is able to facilitate NET‐formation. Cells treated with LL‐37 showed a clear disruption of the nuclear membrane by electron microscopy and confocal immunofluorescence microscopy. Accordingly, when blocking the cleavage and subsequent activation of endogenous LL‐37, NET formation was significantly diminished. Biochemical assays utilizing a random LL‐37‐fragment library indicated that the NET‐induction is mediated by the hydrophobic character of the peptide.

In conclusion, this study demonstrates a novel role of the antimicrobial peptide LL‐37 in host immune defence: the disruption of the nuclear membrane and subsequent formation of NETs.

Grant Funding Source: German Research Council (DFG)
Prolonged functional life span of artificial red cells (Hb‐vesicles) by coexistence of an electron mediator in blood stream (794.7)
Authors: Sakai Hiromi
Published: June 2021

Artificial red cells (Hb‐vesicles, HbV) are one candidate of a transfusion alternative. A purified and concentrated Hb solution is compartmentalized in phospholipid vesicles (liposomes) (Sakai et al., JBC 2008;283:1508‐17). The safety and efficacy of HbV have been examined extensively in preclinical studies. Because of the lack of metHb‐reduction system that is originally present in RBC, HbV gradually increases the metHb level through autoxidation after intravenous injection. In this paper we aimed to prolong the functional life span of HbV by reducing ferric metHb (Fe3+) to ferrous state (Fe2+) by co‐injection of a water‐soluble electron mediator (EM+), and find out the mechanism of in vivo metHb reduction system. HbV was injected intravenously ([Hb] = 10 g/dL, 20 mL/kg) to anesthetized male Wistar rats. After the metHb level of HbV reached to 50%, EM+ was injected intravenously. The metHb level rapidly reduced to around 10%. In vitro experiments of mixing HbV with blood (or washed RBC) and EM+ showed metHb reduction, but not with plasma and EM+. These results indicate that RBC is a source of an electron to reduce EM+ to EMH and it moves across the lipid membranes to pass the electron to metHb in HbV. EM+ effectively mediates electron transfer from RBC to HbV in blood circulation. The detailed mechanism of electron transfer will be presented.

Grant Funding Source: Grant‐in‐aid for Scientific Research from JSPS
GPER‐mediated regulation of nuclear Akt/FOXO3a signaling (795.1)
Authors: Zekas Erin , Prossnitz Eric
Published: June 2021

In addition to its genomic effects, the steroid hormone estrogen has been shown to mediate rapid signaling events in diverse cell types, including breast cancer cells. These effects are, in part, mediated through its canonical soluble nuclear receptor, ERα. However, a novel estrogen receptor known as GPER (previously termed GPR30) has been demonstrated to activate multiple signaling cascades in response to estrogen. We previously established that PI3Kinase‐mediated production of PIP3 in the nucleus is initiated by estrogen‐mediated stimulation of GPER. The mechanism of this activation and its downstream effects are currently not well understood. Here we describe that rapid signaling through GPER activates PI3Kinase leading to the inactivation and translocation of the proapoptotic protein FOXO3a, resulting in its translocation from the nucleus to the cytoplasm. This activation is mediated by both estrogen and the GPER‐selective agonist G‐1, and occurs via the transactivation of EGFR leading to the activation of the p110alpha subunit of PI3K and subsequent AKT‐mediated phosphorylation of FOXO3a. The nuclear exclusion of FOXO3a by GPER activation suggests GPER signaling results in prosurvival cellular effects in breast cancer cells.

Grant Funding Source: NIH
Membrane progesterone receptor alpha: implications for human and mouse preterm labor (795.2)
Authors: Lu Jing , Reese Joshua , Filipovich Yana , Agrawal Varkha , Hirsch Emmet
Published: June 2021

Functional progesterone (P4) withdrawal leading to labor is achieved in part by regulation of the ratio of nuclear progesterone receptor (PR) A to B. The role of membrane‐associated PR (mPR) in pregnancy and labor is not clear. We observed that human placental expression of mPRα mRNA, but not COX2, IL‐1β and oxytocin receptor (OXTR), rises with increasing gestational age at labor. In a mouse model, intrauterine injection of either E. coli or peptidoglycan + polyinosinic: polycytidylic acid on gestational day 15 significantly decreased expression of mPRα in uterus but not in placenta harvested eight hours after injection, and increased expression of COX2 in both uterus and placenta. In a murine macrophage cell line (RAW264.7), activation of mPRs by P4 modified to be active only extracellularly by conjugation to BSA (P4‐BSA, 100 nM) caused a pro‐inflammatory shift in mRNA expression profile, with significant down‐regulation of mPRα and OXTR and up‐regulation of the expression of COX2, IL‐1β, and TNF. Pretreatment with PD98059, a MEK 1/2 inhibitor significantly reduced P4‐BSA‐induced IL‐1β, TNF and COX‐2 mRNA. Inhibition of PKA by H89 blocked P4‐BSA‐induced IL‐1β and TNF mRNA levels. These data suggest that MEK1/2 and PKA activities are involved in the signaling of mPRs. Taken together, we demonstrated that change in mPRα expression and signaling are associated with the inflammatory responses in labor.

Grant Funding Source: R01HD41689
Corticotropin‐releasing factor receptor crosstalk pivotal to ligand‐specific signaling and trafficking (795.3)
Authors: Mahajan Shilpi , Bhargava Aditi
Published: June 2021

Corticotropin‐releasing factor receptors 1 and 2 (CRF1 and CRF2) are GPCRs for the ligands CRF and urocortins (Ucn1‐3). CRF1 and CRF2 activation by their ligands evokes distinct cellular responses, but the signaling and trafficking mechanisms are unclear. In neuronal SK‐N‐MH cells, we show that both CRF receptors are expressed, but only Ucn1 mediates intracellular Ca2+ [Ca2+]i responses, suggesting CRF receptor crosstalk is important in mediating signaling in a context‐dependent manner. Cultured HEK cells expressing CRF1 had a robust [Ca2+]i response to challenge with 100 nM CRF and/or Ucn1, but not Ucn2 or Ucn3. HEK cells expressing CRF2 had a [Ca2+]i response at 10‐100nM doses of Ucn3, but only at high 100nM doses of Ucn1 or Ucn2. Coexpression of CRF1 with CRF2 significantly attenuated CRF and Ucn3‐mediated [Ca2+]i signaling. Cell‐surface labeling of epitope‐tagged receptor showed trafficking to different endosomal compartments after stimulation with a single ligand vs. two simultaneously. By co‐immunoprecipitation and mass spectrometry analysis, we show for the first time that CRF1 and CRF2 physically interact to form functional heterodimers. Tubulin interacts with the CRF receptor complex differently differentially only after stimulation with CRF+Ucn2. Thus, we propose a novel mechanism by which the two CRF receptors physically interact and recruit ancillary proteins in a ligand‐specific manner to regulate signaling.

Grant Funding Source: Supported by NIH NIDDK080787
In vivo and in vitro interaction of calcineurin B homologous protein isoforms 1 and 2 with the Na+ ‐ H+ exchanger isoform 1 (796.10)
Authors: Wallert Clarice , Buchholz Scott , Keifer Lea , Fray Emily , Provost Joseph , Wallert Mark
Published: June 2021

The Na+‐H+ exchanger isoform 1 (NHE1) is a key regulator of cell proliferation, migration and invasion in cells from a variety of solid tumors. The calcineurin B homologous proteins (CHP1 and CHP2) appear to be essential cofactors to support NHE1 function. CHP1 appears to be expressed ubiquitously in healthy tissue, while CHP2 is predominantly expressed in tumor cells. While both CHP isoforms are highly homologous and bind in nearly identical regions on NHE1, each has been identified to have a distinct impact on NHE1 function. In this study we investigate the ability of CHP1 and CHP2 to bind independently and competitively to NHE1. The impact of serum deprivation has been thought to drive CHP2‐NHE1 transport and the effect of low serum on CHP1/CHP2 interaction with NHE1 will be assessed. Using recombinant fusion CHP1, CHP2 and NHE1 proteins a reconstituted‐96 well plate based assay has been developed to quantitate the binding affinity of each CHP isoform for NHE1. We will also demonstrate the competition binding between CHP1 and CHP2 for NHE1 using this in vitro protein‐protein method.
Constitutive mTORC1 activation upregulates catalase and SODs in melanoma cells (796.12)
Authors: Wan Yinsheng , Calianese David , Cammarata Garrett , Best Charles
Published: June 2021

The molecular mechanisms of melanoma’s aggressiveness point to the mutations of EGFR and RAS/RAF and other cellular signaling molecules. Drugs are being developed to target RAS/RAF/MEK/ERK/mTOR signaling in metastatic melanoma. Our studies have demonstrated that anti‐EGFR antibodies do not recognize EGFRs in melanoma cells, suggesting that either mutated EGFR or other EGFR family members exist in melanoma. Oxidative stress induces apoptosis of human skin cells including melanocytes. Antioxidants play important roles to maintain re‐dox status of normal cells. Antioxidases such as catalase and SODs are oxidant scavenger enzymes that modulate the expression and activities of antioxidants. In ovarian cancer cells, we observed that antioxidant NAC potentiates p53 activation. Understanding of antioxidant activity in both melanocytes and melanoma cells will provide insight into disease progression, prognosis, and responses to therapy. Our studies showed that melanoma cells resist to oxidative stress, with constitutive activation of mTORC1 pathway and s6 ribosomal protein phosphorylation, presumably leading to protein synthesis and cell survival. We found also that catalases and SODs are up‐regulated in melanoma cells. Collectively, our data suggest that the constitutive activation of mTORC1 lead to overexpression of catalase and SODs with a result of the resistance to oxidative stress in melanoma.

Grant Funding Source: Supported by NIH
TLR4 signaling regulates PI3 kinase activation and macrophage exophagy (796.16)
Authors: Singh Rajesh , Haka Abigail , Grosheva Inna , Chin Harvey , Maxfield Frederick
Published: June 2021

We have characterized a novel process that macrophages use to degrade species such as aggregated lipoproteins (agLDL) that are too large to be internalized via conventional phagocytosis. Macrophages form an intimate interaction with agLDL through local actin polymerization and form an extracellular compartment to which hydrolytic enzymes are delivered through lysosome exocytosis. The process of extracellular macrophage degradation of large species prior to internalization has been termed “exophagy”. AgLDL degradation by macrophages leads to foam cell formation, a key feature of atherosclerotic plaque development. Therefore unraveling the signaling mechanisms involved in this process may allow therapeutic intervention.

Upon engagement with agLDL, Toll‐like receptor 4 (TLR) interacts with its effector MyD88 and TLR4/MyD88 are critical for lysosome exocytosis and actin polymerization during exophagy. At contact sites with agLDL, PI(3,4,5)P3 is produced, suggestive of class I PI3 kinase activation. Inhibition of PI3 kinase impairs lysosome exocytosis and actin polymerization during exophagy. PI(3,4,5)P3 recruits and activates plekstrin homology (PH) domain containing proteins such as protein kinase B (Akt). AgLDL treatment stimulates PI3 kinase dependent Akt activation, and inhibition of Akt using Akti1/2 impairs lysosome exocytosis and actin polymerization. While PI(3,4,5)P3 levels at contact sites with agLDL peak rapidly (around 20‐30 min) and then decline, the sustained generation of PI(4,5)P2 may act as a lasting signal to promote actin polymerization and lysosome exocytosis at sites of exophagy.

Exophagy may represent a general mechanism, which cells may use to degrade large extracellular species, and there exists a close relationship between this process and various human diseases. Therefore elucidation of signaling mediators regulating these processes would potentially be of widespread importance in a number of diseases other than atherosclerosis.

Grant Funding Source: Supported by NIH
Mutational analysis of calcineurin B homologous protein isoform 2 binding to the Na+ ‐ H+ (796.8)
Authors: Pantera Harrison , Bisnett Brittany , Hasle Daniel , Cottle Wayne , Provost Joseph , Wallert Mark
Published: June 2021

The Na+‐H+ exchanger isoform 1 (NHE1) is a transmembrane protein that regulates a range of cellular functions essential for cancer progression including cell adhesion, proliferation and migration. The calcineurin B homologous proteins (CHP1 and CHP2) appear to be essential cofactors to support NHE1 function. The CHP1 and CHP 2 binding domain on NHE1 is the same, amino acids 515 to 530. CHP2 is expressed primarily in tumor cells where it binds to NHE1 with a 5‐10 fold higher affinity than CHP1. CHP2 expression in tumor cells supports increased invasion and migration. Here we investigate the ability of mutations to a key amino acid in the binding domain (N519) to alter CHP2 binding to NHE1 in vivo. Two distinct site directed mutations to NHE1, N519A and N519D, have been constructed along with the accompanying stable cell lines expressing NHE1 with these mutations. We will present data evaluating CHP2 binding to NHE1 in these cells using a GFP‐CHP construct. We will also evaluate how a change in CHP2 binding alters adhesion, proliferation, and migration in mutant expressing cells as compared to cells expressing wild‐type NHE1.
Insulin‐stimulated PAK1 activation is required for glucose uptake in skeletal muscle cells (797.1)
Authors: Tunduguru Ragadeepthi , Chiu Tim , Klip Amira , Thurmond Debbie
Published: June 2021

Skeletal muscle accounts for ~80% of postprandial glucose clearance, crucial for maintaining euglycemia and insulin sensitivity. Insulin‐stimulated glucose uptake entails recruitment of glucose transporters (GLUT4) to the plasma membrane (PM) in a process that requires cortical F‐actin remodeling; this process is dysregulated in Type 2 Diabetes. Recent studies have implicated PAK1 as a required element in GLUT4 recruitment in mouse skeletal muscle in vivo, although its underlying mechanism of action and requirement in glucose uptake remains undetermined. Toward this, we have employed the PAK1 inhibitor, IPA3, in studies using L6‐GLUT4‐myc muscle cells, the premiere skeletal muscle cell line for glucose uptake analyses. Confocal microscopy showed IPA3 to fully ablate insulin‐stimulated GLUT4 translocation to the PM, corroborating the observation of ablated insulin‐stimulated GLUT4 accumulation in the PM of skeletal muscle from PAK1‐/‐ knockout mice. IPA3‐treatment also abolished insulin‐stimulated glucose uptake into skeletal myotubes. Mechanistically, live‐cell imaging of myoblasts expressing the F‐actin biosensor LifeAct‐GFP treated with IPA3 showed blunting of the normal insulin‐induced cortical actin remodeling. This blunting was underpinned by a loss of normal insulin‐stimulated Cofilin dephosphorylation in IPA3‐treated myoblasts, suggesting a new model: insulin activates PAK1 to signal downstream to Cofilin to trigger actin severing, providing substrate for F‐actin remodeling, which facilitates GLUT4 vesicle translocation toward the PM where GLUT4 promotes glucose uptake into the skeletal muscle cell.

Grant Funding Source: DK077614
RUNX2 O‐GlcNAc modification links osteogenesis and nutrient metabolism in bone marrow mesenchymal stem cells (800.3)
Authors: Nagel Alexis , Ball Lauren
Published: June 2021

Decreased bone mineral density and increased risk of fracture in patients with Type I and II diabetes suggest that bone is a nutrient‐responsive tissue, however nutrient‐responsive mechanisms which modulate bone formation are not well understood. We previously identified multiple osteoblast proteins modified by O‐linked N‐acetylglucosamine (O‐GlcNAc), a post‐translational mechanism which modulates nutrient‐induced transcriptional and epigenetic programs. In this study we elucidate novel sites of O‐GlcNAc modification on runt‐related transcription factor 2 (RUNX2), the master osteogenic transcriptional regulator, using C18 nLC‐MS/MS and HexNAc product ion‐triggered ETD fragmentation. Sites of RUNX2 HexNAc modification reside within known PST‐ and N‐terminal activation domains. In addition to HexNAc modification, we observe acetylation and methylation of RUNX2 as well. O‐GlcNAc modification of endogenous RUNX2 was confirmed in bone marrow mesenchymal stem cells (BMMSCs) by click‐chemistry based chemoenzymatic labeling. Global elevation of protein O‐GlcNAc by Thiamet G (20 µM) inhibition of O‐GlcNAcase (OGA) significantly enhanced activity (65.8% over vehicle; n=3) and transcript expression (24.3% over vehicle; n=2) of the RUNX2‐target alkaline phosphatase (ALP) in osteogenic BMMSCs after 6 days. Addition of dexamethasone and rhBMP2/7 to the differentiation media resulted in decreased OGA activity (70.3% over vehicle; n=3) after 8 days. These data suggest that OGA and OGT modulate early osteogenic pathways in developing osteoblasts which may be partly mediated through glycosylation of RUNX2. Ongoing studies focus on determining the effect of RUNX2 O‐GlcNAcylation on transcriptional activity and protein interactions as they contribute to the regulation of osteogenesis.

Grant Funding Source: RO1‐DE020925‐01A1
Inositolless death and the lack of progression through the mitotic cell cycle to anaphase in S. cerevisiae (801.4)
Authors: Hanson Barbara , O’Connor Paul , Frandina John , Pittari Joseph , Munezero Jean , Croglio Vincent
Published: June 2021

When an inositol mutant (MC6A) of S. cerevisiae is starved for inositol, the cells undergo cellular changes that result in the loss of viability known as inositolless death. One target of inositolless death in S. cerevisiae may be the mitotic cell cycle because of the accumulation of polymerized tubulin in these cells. Normally, tubulin starts to depolymerize during anaphase of the cell cycle. One explanation for polymerized tubulin is that the cells enter the cell cycle but do not progress through anaphase where this would occur. To answer this question the levels of mRNA for the genes: Securin (Pds1), Separin (Esp1) and a major subunit (Apc1) of the anaphase‐promoting complex, were measured using RT‐PCR with RNA extracted from cells grown in the presence and absence of inositol for 0, 3, and 24 h. The mRNA levels for Pds1 and Apc1 declined to 57% and 55% of control levels at 3 h and 27% and 33% at 24h. The mRNA levels for Esp1 declined the most rapidly to 22% of control levels at 3h and to 20% at 24h (Fig. 5). The rapid reduction in Esp1 mRNAs and the resulting low levels of the Esp1p protease may prevent the separation of sister chromatids. This would prevent the start of anaphase and the resulting spindle microtubule depolymerization.

Grant Funding Source: Canisius College
Deciphering crucial threonines towards functionality of an essential eukaryotic‐type Ser/Thr kinase from M. tuberculosis (802.1)
Authors: Ravala Sandeep , Yadav Ghanshyam , Chakraborti Pradip
Published: June 2021

PknA is an essential Ser/Thr kinase in mycobacteria and is involved in several physiological processes including cell division and cell wall biosynthesis. Previous results of phospho‐amino acid analysis through western blotting established that PknA predominantly phosphorylates at threonines. Structurally, PknA comprises of four domains, spanning catalytic, juxtamembrane, transmembrane and extracellular domains. Unlike other eukaryotic‐type Ser/thr kinases in M. tuberculosis, PknA catalytic domain was found to be devoid of autophosphorylating ability and only in conjunction with juxtamembrane, it exhibited catalytic activity. Therefore, the focus of the study was to delineate the minimum region of juxtamembrane required for PknA function so that crucial threonines involved in its phosphorylation could be deciphered. Using mutational analysis and E.coli based phenotypic assay as tools, we report here the essentiality of six residues from juxtamembrane region for the kinase activity exhibited by PknA. Furthermore, we observed Thr‐180 and Thr‐224 along with the activation loop threonines were critical for the functionality of PknA.

Grant Funding Source: Supported by CSIR Network Project
Regulation of cell signaling: GRK2 dependent phosphorylation of alpha2A‐adrenergic receptors (802.12)
Authors: Hebert Aaron , Sterne‐Marr Rachel
Published: June 2021

Cells relay information, such as the fight or flight response, via chemicals, such as adrenalin, and one of two classes of adrenergic receptors, α and β. The α2A‐adrenergic receptor (α2AAR), has been shown to play a key role in vasoconstriction in some parts of the cardiovascular system, and is the target of pharmaceuticals. Proper regulation of cellular signals by desensitization of the receptor is essential to maintaining the organismal status quo or homeostasis. Phosphorylation, or the addition of a highly charged phosphate group, is carried out by kinases and is a common mechanism of receptor desensitization. We study a kinase, called GRK2, that phosphorylates adrenergic receptors after they are treated with adrenalin. One way to study the phosphorylation of the α2AAR in intact cells is to use antibodies that specifically recognize the phosphorylated receptor. Because no commercially available α2AAR phosphosite antibodies currently exist, we set out to generate such a reagent. A functional phosphosite antibody that detects α2AAR phosphorylation was isolated and characterized. This antibody that we call Rb617 was used to test whether the phosphorylation of α2AAR in intact cells was adrenalin‐induced and GRK2‐dependent in primate cells in culture. We found that the α2AAR was phosphorylated in the absence of adrenalin and exogenous GRK2 suggesting that, in addition to GRK2, other cellular kinases modify the α2AAR. Further intact cell phosphorylation assays will likely include the use of inhibitors to minimize the effect of phosphorylation due to these other kinases so that we might study phosphorylation by GRK2.
Quaternary architecture of PKA RIβ holoenzyme reveals isoform diversity (802.5)
Authors: Ilouz Ronit , Wu Jian , Kornev Alexandr , Taylor Susan , Bubis José , Blumenthal Donald
Published: June 2021

The cAMP‐dependent protein kinase A (PKA) holoenzyme is composed of two catalytic (C) subunits and a regulatory (R) subunit dimer. Although much is known about the structures and function of RIα, RIIα and RIIβ isoforms, little is known about RIβ. Given its unique biological importance and functional non‐redundancy, we hypothesized that RIβ will also have unique structural properties. We show first that the RIβ2:C2 overall quaternary structure is different from RIα2:C2, its closest homolog, using small angle x‐ray scattering (SAXS). We found that RIβ is more enriched in mouse mitochondria than RIα. We then obtained a crystal structure of the tetramer holoenzyme with the full‐length RIβ bound to the C‐subunit. This is the first PKA tetramer structure where all the domains can be visualized. The head‐to‐head alignment of the C‐subunits is in striking contrast to RIα where the C‐subunits do not contact each other. The structure allows us to appreciate how assembly of the R2:C2 complex creates isoform diversity even though the conformations of the R:C heterodimers are similar in all isoforms. Conserved tertiary structure does not translate into conserved quaternary structure. This structure paves the way for exploring the biological properties of RIβ.
A proteomic approach identifies plasma proteins correlated with vitamin B6 in Nepalese children (804.14)
Authors: Yakub Mohsin , Schulze Kerry , Cole Robert , Christian Parul , Betz Joshua , Ruczinski Ingo , Yager James , Wu Lee , Groopman John , West Keith
Published: June 2021

Vitamin B6 is a coenzyme for >140 distinct enzymes involved in amino acid metabolism. In the plains of Nepal, we have reported 43% of children age 6‐8 years of age to be B6 deficient (pyridoxal‐5’‐phosphate, PLP <20nmol/L), revealing a public health problem. This study identified differentially abundant plasma proteins in relation to B6 status in 500 6‐8 yr old Nepalese children that represent possible B6 biomarkers with the potential to predict B6 status. Plasma PLP concentration was analyzed by HPLC, and relative abundance of plasma proteins by isobaric tags for relative and absolute quantification (iTRAQ) tandem mass spectrometry. A total of 87 proteins were associated with PLP (for all, q<0.1, p<0.008), defining a plasma B6 proteome: 39 and 48 were positively and negatively correlated (r from 0.51 to 0.29 and ‐0.56 to ‐0.21) with PLP, respectively. Identified proteins are involved in numerous metabolic pathways, including cell proliferation, proteolysis, adhesion, acute phase reaction, coagulation and transport. Linear mixed effects analysis estimated ~60% of the variance (R²) in plasma PLP could be estimated by the relative abundance of 5 proteins (complement 9, cyclin D‐type binding‐protein 1, dynein, axonemal, heavy chain 10, leucine rich repeat containing 47, and NOP2/Sun domain family, member 6). A predictive plasma PLP proteome may exist for assessing population B6 status.

Grant Funding Source: Supported by Bill and Melinda Gates Foundation Grants 614 and 5241
Effects of two micronutrient‐fortified food aid products containing different levels of dairy protein on anthropometric variables in rural pre‐school children in Guinea‐Bissau (804.15)
Authors: Batra Payal , Schlossman Nina , Balan Ela , Pruzensky William , Saltzman Edward , Roberts Susan
Published: June 2021

Food insecurity is widespread in Guinea‐Bissau. We tested the efficacy of two Micronutrient‐Rich Ready to Use Supplementary Foods (MNR‐RUSF) in preschool children aged 3‐5 years over 3 months. 9 preschools (n=421 children) were randomly assigned to one of two interventions (MNR‐RUSF sachets containing 500 kcal with either 15% or 33% of protein from dairy sources, provided 5 mornings/week) or to a control (C) group that received no MNR‐RUSF. Weight, height and mid upper arm circumference (MUAC) were measured at baseline and endline. At baseline, 5.6% of enrollees had weight‐for‐age z scores <‐2 and 7.1% had height‐for‐age z scores <‐2. Both intervention groups had a significant increase in weight over time vs. controls (15% group = 0.77+0.75 kg, 33% group = 0.76+0.78 kg, C = 0.42+0.1.8 kg, p<0.05) but not in height (p=0.34) and changes in these variables were not different between the 15% and 33% groups (p= 0.99 and p=0.38). There was a significant increase in MUAC in both the intervention groups relative to controls, and the 33% group had a greater increase than the 15% group (15% group = ‐0.03±0.68 cm, 33% group = 0.14±0.78 cm, C = ‐0.31±0.67cm, p<0.001). This study is the first to focus on anthropometry in preschool children in Guinea‐Bissau and indicates that, while both standard and high‐dairy MNR‐RUSF products improve weight in preschool populations with a high prevalence of malnutrition, those containing high levels of dairy protein have additional benefits for MUAC.

Grant Funding Source: Supported by USDA/FAS under Agreement FFE‐657‐2012/043‐00
Plasma proteins and vitamin K (PIVKA‐II) status in school aged children in Nepal (804.22)
Authors: Lee Sun Eun , West Keith , Cole Robert , Schulze Kerry , Ruczinski Ingo , Betz Joshua , Yager James , Groopman John , Shrestha Sudeep , Christian Parul
Published: June 2021

Vitamin K deficiency (VKD) is known to disrupt blood coagulation. However, broader public health effects of childhood VKD are unknown. We explored the plasma proteome to identify proteins that co‐vary with VK status and that may be associated with VKD in Nepalese children. We measured abundance of plasma proteins using quantitative mass spectrometry and concentration of protein induced by VK absence (PIVKA‐II), an indicator of VK status, in 500 rural children 6‐8 y of age. An elevated PIVKA‐II concentration (>2ng/mL), reflecting VKD and observed in 25% of children, was associated with higher concentrations of low‐density lipoproteins, total cholesterol, and triglycerides, and greater prevalence of vitamin D, E (cholesterol adjusted), and B12 deficiencies (all p<0.05). Among ~980 proteins observed in >10% of children, prostaglandin E synthase 3, coagulation factor II, vascular endothelial cadherin, voltage‐dependent calcium channel, gelsolin, and hemoglobin‐delta abundance were correlated with PIVKA‐II (all p<0.001, q<0.1). With a relaxed discovery threshold, other hemoglobin chains, glycolysis enzymes of erythrocytes and antioxidants were positively correlated with PIVKA‐II (all p<0.02, q<0.4). Since these proteins are involved in synthesis of vasodilators, regulation of Ca2+ influx in smooth muscle cells, and red blood cell metabolism in response to hypoxia, subclinical VKD may be involved in vascular and erythrocyte adaptation to low circulating oxygen in blood.

Grant Funding Source: Supported by the Bill and Melinda Gates Foundation (Grant 5241).
A brief survey of digital media access among pregnant teens (806.12)
Authors: Whisner Corrie , Cooper Elizabeth , Pressman Eva , O'Brien Kimberly
Published: June 2021

Adolescents have greater access to accurate and relevant health information via the internet. The aim of this study was to briefly describe the availability and use of digital media among pregnant teens. A total of 32 girls (aged 13‐18) between 12 and 30 weeks of gestation enrolled in an intervention to assess the effects of vitamin D supplementation on inflammation across pregnancy. Volunteers completed a short survey on internet use, participation in social media and interest in learning about prenatal care. Teens were racially diverse (48.5% Black, 24.2% White, 24.2% other, and 3.0% American Indian) and 48.5% of teens were enrolled in WIC. All but one teen had internet access with the majority of teens using the internet on home (43.8%, n=14) or library (15.6%, n=5) computers. More than half (59.4%) of the teens used the internet daily while 15.6% and 18.7% of teens accessed the internet one to a few times per week and a few times per month or less, respectively. Digital communication was popular such that 71.9% (n=23), 54.8% (n=17) and 65.6% (n=21) of teens reported the use of Facebook, email and personal cell phones, respectively. Half (n=16) of the teens with phones had cellular internet access and all had phones with text messaging. When asked if teens would like to receive information to promote healthy pregnancies, 87.5% (n=28) of respondents said yes. Preferences for receiving this information were 53.1%, 18.8%, 18.8% and 3.1% for text messages, paper, email and Facebook, respectively. Pregnant teens value digital media and leveraging the use of social media and internet resources may be affordable and effective ways of informing this high risk group about prenatal care.

Grant Funding Source: Supported by AFRI Grant No. 2012‐67017‐30216 and 2012‐67012‐19815
Examining middle school children's willingness to consume energy‐dense foods and sweetened beverages (808.16)
Authors: Zawodniak Brittany , Cater Melissa , McGuerty Amber , Prinyawiwatkul Witoon , Tuuri Georgianna
Published: June 2021

It is important to understand children’s willingness to consume foods and beverages because preferences impact behavior change. This study examined children’s willingness to eat energy‐dense foods, energy‐dilute foods, sweetened beverages and unsweetened beverages. 312 children attending 4‐H summer camp were recruited to participate. 307 provided their gender and grade and 305 provided their race/ethnicity. Children were in the following grades: 4th, n = 71 (23.1%), 5th, n = 130 (42.3%), and 6th, n = 106, and 184 were female (59.9%). 251 children (82.3%) were white, 41 were black (13.4%), 13 were from other racial groups. Surveys were distributed by one trained investigator using a standard protocol. Student willingness data gathered from a 7‐point Likert‐type scale were collapsed into three categories: 1 = unwilling, 2 = neither unwilling nor willing and 3 = willing. Race/ethnicity was categorized as white or non‐white. Mean scores were compared using the Welch’s F test. Males were more willing than females (F = 4.846; p = 0.03) and white children were more willing than non‐white (F = 4.489; p = 0.04) to consume energy‐dense foods. Differences in willingness to consume items in other food/beverage groups were not observed. This study provides valuable insight into food preference patterns, and therefore eating behaviors, of children for the purpose of future nutrition interventions.
Ideas to solve the obesity epidemic: a pilot study evaluating college students’ perspectives (808.22)
Authors: Fernandes Nicolle , Trivedi Rupal , Desouza Josephine
Published: June 2021

As the obesity crisis encompasses the younger generation, this study aimed at evaluating college students’ perspective on the obesity solution. Students enrolled in a junior‐level nutrition class at a Midwestern university participated in an online discussion that solicited ideas (open‐ended format) to solve the obesity problem. Discussions were hosted on the university’s Blackboard Learning System. Seventy‐eight college students (69 females and 16 males); mean age of 20 + 3.0 years and average body mass index (BMI) of 24.7 + 4.7 were included. More than half the participants identified nutrition education 65% (n=51) and physical activity 55% (n=42) as key aspects in managing obesity. Within the education domain, emphasis was placed on the value of parents and children taking nutrition classes mutually, which might help to carry the family unit to a healthier body weight. The theme of family involvement also extended towards engagement in physical activity where family bonding was deemed an additional benefit. Approximately 36% (n=28) of students believe that parents play an influential role in controlling obesity in children. Parents may leverage that control by limiting for example, youngster’s screen time. Additionally, parents can positively influence children by leading through personal examples of good nutrition and exercise routines. Students also acknowledged the media’s influence on food choices. Other ideas included structuring the grocery store for optimum display of healthier foods, regulation of cafeteria menus, making sports less competitive and more fun, and encouraging the use of nutrition labels. Additionally, providing a tax break for healthy foods at the expense of unhealthy foods was suggested. In conclusion, although college students, on average, recognize the importance of the role of nutrition education and physical activity in managing the obesity epidemic, they understand the complex nature of the problem and believe that the solution may start at “home”.

Grant Funding Source: None
Dietary supplement research portfolio at the National Institutes of Health (NIH) (809.1)
Authors: Garcia‐Cazarin Mary , Wambogo Edwina , Regan Karen , Davis Cindy
Published: June 2021

The U.S. dietary supplement market increased 7.5% in 2012 compared to 2011, reaching $32.5 billion in sales. Therefore, federally supported research on dietary supplements is important to determine their health effects, safety and efficacy. A portfolio analysis was performed across the NIH and the Office of Dietary Supplements (ODS) for the fiscal years 2009‐2011, using the databases Human Nutrition Research information Management system (HNRIM) and Computer Access to Research on Dietary Supplements (CARDS). The results indicated that total NIH dietary supplement‐related funding for FY 2009‐2011was $855M ($295M in 2009, $311M in 2010, and $249M in 2011). The institutes and centers with the highest investment in dietary supplement research were: NHLBI ($135M), NCI ($188M), NCCAM ($99M), NIDDK ($68M), NIEHS ($58M), and ODS ($32M). The dietary supplement ingredients receiving the most funding were: botanicals (22%), vitamins (20%), lipids (14%), and minerals and trace elements (10%). The top three outcome research areas were: cancer (61% of total dietary supplement investment), cardiovascular disease (47%), and women’s reproductive health (38%). In FY 2009, ODS provided 3.5% of the NIH investment in dietary supplement research, 3.6% in 2010, and 4.1 % in 2011. ODS funding focused on cellular, enzymatic, or molecular mechanisms (64% of total ODS funding). This portfolio analysis demonstrates that NIH has committed significant funding to dietary supplement research in an effort to expand the scientific knowledge base in the efficacy and safety of dietary supplements.
Biomaterial scaffolds in regenerative medicine (81.2)
Authors: Eliseeff Jennifer
Published: June 2021

Regenerative medicine and tissue engineering strategies aim to provide tissue substitutes for repair of defects due to disease, trauma, or congenital abnormalities. Biomaterials play a key role in design strategies in tissue engineering functioning to provide a three dimensional environment for cells, present biological cues, and implant in a physiological milieu. Basic research topics including stem cell biology, small molecule therapeutics, and metabolism in combination with biomaterials research in regenerative medicine will be discussed. Finally, translation of key technologies to clinical application in musculoskeletal and soft tissue reconstruction will be presented.