bluuebunny/crossref_metadata_2025_split huggingface dataset:
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
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
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
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).