bluuebunny/crossref_metadata_2025_split huggingface dataset:
ER stress dictates inflammatory, but not hormonal, lipolytic triggers in adipocytes
Authors: Schertzer Jonathan D. , Foley Kevin , Duggan Brittany , Heal Mark , Chi Wendy , Barra Nicole 
Published: June 2021

ObjectiveObesity is characterized by inflammation that can impair endocrine control of cell metabolism. The triggers and stress responses of these immune‐mediated defects are ill‐defined. There is a reciprocal relationship between inflammation and lipolysis in adipocytes, but hormones and adrenergic signals can also stimulate lipolytic programs in adipocytes. This study aimed to identify the cell stress responses that differentiate inflammatory and hormonal triggers of lipolysis in adipocytes.MethodsLipolysis was measured by glycerol release from 3T3‐L1 adipocytes treated with inhibitors of ER stress, inositol‐requiring protein 1α (IRE1α) or tyrosine kinases before stimulation with inflammatory lypolytic stimuli. The inflammatory stimuli included bacterial peptidoglycan, lipopolysaccharide, and tumor necrosis factor. The Hormonal or adrenergic stimuli tested included isoproterenol and forskolin. Inflammation was characterized by Il6 secretion and NF‐κβ activity.ResultsInhibition of the kinase activity of IRE1α was sufficient to block lipolysis and Il6 secretion caused by thapsigargin‐induced ER‐stress in adipocytes. Inhibition of IRE1α kinase activity blocked augmented lipolysis from inflammatory, but not hormonal stimuli. Inhibition of IRE1α also blocked augmented Il6 secretion from all inflammatory stimuli. Inhibition of IRE1α blocked the increased NF‐κB activity from all inflammatory stimuli except for lipopolysaccharide. Inhibition of ABL kinases with imatinib did not alter lipolysis. Inhibition of IRE1α RNase activity did not alter lipolysis. The potent RIPK2 inhibitor ponatinib blocked lipolysis, Il6 secretion, and NF‐κβ activation stimulated by peptidoglycan, but did not alter lipolysis from other inflammatory stimuli, despite attenuated Il6 secretion.ConclusionsThe ER stress sensor IRE1α is essential for inflammation‐induced lipolysis and Il6 secretion in adipocytes. Neither RIPK2 nor NF‐κβ can fully capture the IRE1α lipolyitic pathway from inflammatory stimuli. These findings show that IRE1α discriminates inflammation‐induced lipolysis linked to ER stress from hormonal or adrenergic triggers of adipocyte lipolysis.Support or Funding InformationThis research is supported by operating grants to JDS from the Natural Sciences and Engineering Research Council (NSERC). JDS held CDA Scholar (SC‐5‐12‐3891‐JS) and CIHR New Investigator awards (MSH‐136665) and holds a Canada Research Chair in Metabolic Inflammation. BD was supported by an Ontario Graduate Scholarship (OGS). KF is supported by an NSERC postdoctoral fellowship.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeting the Prolactin Receptor Signaling Using an Antipsychotic Drug to Suppress Pancreatic Cancer
Authors: Dandawate Prasad , Kaushik Gaurav , Subramaniam Dharmalingam , Ramamoorthy Prabhu , Ghosh Chandrayee , Choudhury Sonali , Standing David , Dhar Animesh , Thomas Sufi M. , Santimukul Santra , Padhye Subhash , Tawfik Ossama , Weir Scott , Jensen Roy A. , Anant Shrikant 
Published: June 2021

Pancreatic cancer (PCa) is deadly cancer with an abysmal five‐year survival rate of < 6%. PCa has a bad prognosis and is associated with resistance to standard of care chemotherapeutic gemcitabine and high recurrence rate after surgery. Unfortunately, gemcitabine remains the drug of choice for metastatic pancreatic adenocarcinoma, with minor improvements in survival rate. Hence, there is a need for novel therapeutic interventions for the treatment of PCa. Here we report that prolactin receptor (PRLR) is overexpressed in both PCa tissues and in cell lines. When it is bound by the ligand prolactin (PRL), various downstream events such as JAK‐STAT and ERK‐MAPK signaling pathways are activated. In PCa cell lines, PRL treatment induced dose‐ and time‐dependent STAT3, and ERK1/2 phosphorylation. Moreover, while PRL did not affect PCa cell proliferation, it significantly enhanced pancosphere formation suggesting that PRL affects stem cells. We manipulated PRLR levels using the CRISPR/Cas9 system. This resulted in the lower colony and spheroid formation capacity, decreased migration in vitro as well as reduced tumor‐forming capacity in an orthotopic model of PCa in C57BL/6 mice. Based on these results, we propose that PRL signaling is a novel target for therapeutic intervention. Currently, there are no known small molecules targeting PRL‐PRLR signaling, and X‐ray crystal structure of the intracellular domain of PRLR is not available. Hence, we obtained a homology model for the C‐terminal intracellular region of the receptor and performed a virtual screening for small molecules in silico. In this, we demonstrated that penfluridol (a first‐generation antipsychotic drug) interacts with the JAK2 binding site of the receptor. Penfluridol is known for its long half‐life and is used in the treatment of chronic schizophrenia. We experimentally confirmed binding of penfluridol to PRLR in the cells by using the cellular thermal shift assay (CETSA). Penfluridol treatment decreased PRL‐induced STAT3 and ERK phosphorylation, spheroid formation as well as the migration of PCa cells. We observed a significant increase in autophagy based on increased levels of LC3B, p62 and enhanced expression of autophagy‐related genes ATG‐5, −7 and −12 after penfluridol treatment. Penfluridol treatment also significantly decreased tumor burden in orthotopic PCa model and induced the expression of autophagic related proteins. Taken together, these results suggest that PRL‐PRLR signaling is important for PCa growth and repurposing penfluridol to target PRL‐signaling an effective therapeutic targeting strategy for treating the disease.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparison of Isolation, Characterization and Differentiation of Human Umbilical Cord Mesenchymal Stromal Cells in a traditional 2D culture versus a 3D culture.
Authors: Jirakittisonthon Thitikan , Murnane Joan , Snyder Larry , Cromer Adrienne , Weiss Mark 
Published: June 2021

Mesenchymal stromal cells (MSCs) are attractive candidates for regenerative medicine since they appear to be safe and effective for modulating diseases with an inflammatory component such as osteoarthritis and Crohn's disease. The immune modulating and paracrine effects noted clinically cannot be explained solely by cellular engraftment since a fraction of the injected MSCs can be recovered one month after injection. Traditional two‐dimensional (2‐D) MSC culture evaluates cells expanding along a flat surface. In contrast, their natural environment, e.g., in vivo, is complex and three‐dimensional (3‐D). We speculate that 3‐D cell culture may more accurately reflect in vivo cellular behavior. The objective of this study was to characterize human umbilical cord‐MSCs cultured in a traditional 2‐D on tissue culture plastic versus a 3‐D hydrogel model. We compared expression of cell surface receptors (Flow Cytometry), metabolic activity (MTT Assay), and histological cellular differentiation of HUC‐MSCs isolated from 2‐D culture conditions to 3‐D hydrogels. We found trilineage differentiation potential and mesenchymal surface markers similar to that observed in 2D culture. This pilot work, together with published spheroid MSC culture work, suggests that surface interactions and cell‐cell interactions brought about by 3D culture influences cell physiology but doesn't interfere with the cells meeting the ISCT minimal MSC definition.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Probiotic Metabolites Promote Stem Cell Proliferation and Differentiation
Authors: Wu Danielle , Fu Richard , Scoffield Jessica , Kannappan Ramaswamy 
Published: June 2021

Probiotic metabolites (PM) impart numerous health benefits in human and may have a potential to induce cell proliferation and differentiation. We tested the effects of PM released by probiotics found in yogurts on stem cell proliferation and differentiation. Five different brands of yogurts (Oikos, Stonyfield, Yoplait, Chobani and Activia) often sold in the supermarkets were tested in this study. Yogurt cultures analyzed by Gram‐staining and DNA sequencing of 16s rRNA gene identified that these yogurts are not equally supplemented and include different types and various amounts of probiotics. The probiotics were cultured and the condition media containing PMs were collected and used in our study. To evaluate the effect of PM on stem cells, human induced pluripotent stem cells (iPSC) were treated with probiotic conditioned media. We found that probiotic Lactobacillus reuteri conditioned media (LRCM) induced iPSC proliferation at lower doses but inhibited cell proliferation at higher doses. Additionally, expression of the proliferating cell nuclear antigen (PCNA) and pluripotent stem cell factors, OCT4, SOX2 and NANOG transcripts was upregulated in iPSCs treated with LRCM. iPS cells exposed to LRCM during in vitro differentiation led to enhanced expression of myocyte markers, Nkx2.5 and cTnT. Consistently, sodium butyrate, a defined bacterial metabolite (short chain fatty acid), had similar effect on iPSC proliferation and expression of pluripotent markers and myocyte markers. In summary, we have demonstrated that probiotics in different brands of yogurts are diverse in their numbers, viability and composition. Further, our results show that PMs can potentiate iPS proliferation and induce cardiomyocyte differentiation. Our findings have laid a foundation for further investigation of how probiotics and its metabolites from yogurt affect human health and disease.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Doxorubicin‐induced fetal glomerular mesangial cell apoptosis involves NADPH oxidase‐dependent reactive oxygen species generation
Authors: Matthews Anberitha T. , Soni Hitesh , Buddington Randal K. , Adebiyi Adebowale 
Published: June 2021

Animal studies have shown that chemotherapeutic agent doxorubicin (DOX) causes fetal toxicity, including renal abnormalities. Although DOX‐associated nephropathy includes oxyradical stress and glomerular injury, its effect on fetal glomerular cells remains unclear. This study aimed to examine the effect of DOX on fetal glomerular mesangial cell (FE‐GMC) survival. Furthermore, we tested the hypothesis that NADPH oxidase (NOX)‐dependent reactive oxygen species generation contributes to DOX‐induced alterations in FE‐GMCs. Confocal microscopy and high‐performance liquid chromatography revealed that the level of 2‐hydroxyethidium was increased in DOX‐treated cells when compared to DMSO controls, which was reversed by selective NOX inhibitor, apocynin. Apocynin also attenuated DOX‐induced caspase 3/7 activation in FE‐GMCs. These data suggest that DOX induces apoptosis in porcine FE‐GMCs by promoting NOX‐dependent oxidative stress. Caspase‐mediated cell death may contribute to the toxic effects of DOX on the developing kidney.Support or Funding InformationR01DK101668; R01DK101668‐04S1This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nicotine Attenuates Acetate‐Induced Increase of Cytosolic Reactive Oxygen Species in Dopaminergic‐Like PC12 Cells
Authors: Behnke Jessica E. , Chapp Andrew D. , Shan Zhiying , Chen Qing‐Hui 
Published: June 2021

We have previously reported that the major ethanol metabolite, acetate (6 mM), significantly (p<0.05) increases levels of cytosolic calcium in neural growth factor (NGF) derived PC12 cells measured in real time with Fluo‐4AM. Literature suggests that increased intracellular calcium through glutamatergic channels binds with calmodulin to trigger a cascade leading to the formation of nitric oxide (NO), which can contribute to cell death. We therefore hypothesized that treatment of PC12 cells with acetate (6 mM) would elicit an increase in the levels of cytosolic reactive oxygen species (ROS). Furthermore, it has been shown that nicotine induces protection against glutamate cytotoxicity, although the mechanism is still not fully understood. NGF derived PC12 cells exposed to acetate (6 mM) for 4 hrs had a significant (p<0.05) increase in ROS positive staining cells relative to control (44.3 ± 1.8 % (N=5) vs 33.3 ± 1.5 % (N=3)), measured using flow cytometry with an oxidative stress detection probe (CellROX Orange, Thermofisher). Co‐application of acetate (6 mM) and nicotine (10 μM) was able to significantly (p<0.05) attenuate the acetate‐induced increase in cytosolic ROS back to baseline levels. Percent cells with positive staining are: control (28.6 ± 2.1 %, N=3), acetate (6 mM) (47.5 ± 5.4 %, N=3) and acetate (6 mM) and nicotine (10 μM) (24.04 ± 3.8 %, N=5). This data suggests that nicotine's neuroprotective effect on glutamatergic cytotoxicity may be due, at least partially, to nicotine's ability to reduce the amount of NO formed intracellularly.Support or Funding InformationFunding support: AHA 16PRE27780121, Andrew Chapp, R15‐HL129213, Zhiying Shan; R15‐HL122952, Qinghui ChenThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of Monocyte Age on Influenza A Virus‐induced Lung Injury
Authors: Coates Bria , Turner Margaret , Ciesielski Mark , Cheng Yuan , Ridge Karen 
Published: June 2021

RationaleInfluenza A virus (IAV) causes more life‐threatening infections in healthy children than in healthy adults. In our mouse model of juvenile IAV infection, juvenile mice clear the virus at the same rate as adult mice, but develop more severe lung injury and have increased mortality compared to adult mice. We previously found that the monocytes recruited to the lungs during IAV infection exhibited a more inflammatory phenotype in juvenile mice than in adult mice. These juvenile monocytes exacerbated IAV‐induced lung injury in juvenile mice, and inhibition of their recruitment protected juvenile mice from IAV‐induced lung injury without impacting viral clearance. Based on these findings, we hypothesized that juvenile monocytes contribute to excessive inflammation and lung injury during IAV infection, and that adult monocytes could protect juvenile mice from IAV‐induced lung injury and death.MethodsPregnant mice were treated with busulfan (15 mg/kg i.p. day E18 and E19) to cause myelosuppression in fetal pups. Following birth, additional busulfan was given to juvenile pups (10 mg/kg i.p. day of life 1). Bone marrow derived stem cells were harvested from adult (10 week old) donor mice bearing the CD45.1 marker and injected into the myelosuppressed recipient pups (CD45.2) on day 2 of life (7.5 × 106 cells intravenously). Four weeks following transplantation, juvenile recipient mice were then infected with IAV (strain WSN, 25 PFU, intratracheally) and monitored for the development of lung injury and survival.ResultsJuvenile mice expressing juvenile CD45.2 cells had a median survival of 9 dpi. In contrast, 90% of the juvenile mice expressing adult CD45.1 were alive at 9 dpi and the median survival was >62%. Histological examination of the lungs showed decreased lung injury in IAV‐infected juvenile mice expressing adult CD45.1, as compared to control mice. Juvenile mice that recruited adult monocytes to the lungs during IAV infection had decreased lung injury compared to juvenile mice that recruited juvenile monocytes to the lungs during IAV infection.ConclusionsJuvenile mice are more susceptible to IAV‐induced lung injury and death than adult mice. Recruitment of juvenile monocytes to the lungs during IAV infection exacerbates inflammation and worsens outcomes. Transplantation of adult monocytes into juvenile mice lessens lung injury and improves survival. This suggests that juvenile monocytes are particularly injurious in juvenile IAV infection and may have age‐related cell‐intrinsic properties that exacerbate lung injury.Support or Funding InformationPediatric Critical Care and Trauma Scientist Development Program (NICHD K12)American Thoracic Society/American Lung AssociationNHLBIThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Measurement and Analysis of the Mitochondrial Calcium Sequestration and Effects on the Buffering System
Authors: Strubbe Jasiel O. , Zhang Yizhu , Truong Hai , Bazil Jason N. 
Published: June 2021

Calcium is a key signaling molecule found to regulate many cellular processes, including but not limited to, cellular physiology, bioenergetics, transcriptional events, signal transduction, and reactive oxygen species (ROS) production. While mitochondrial calcium dynamics have been extensively studied, it is yet unclear how calcium is sequestered and buffered in a quantitatively and mechanistic manner. Mitochondrial‐calcium homeostasis plays a crucial role in cell fate by a tightly‐regulated buffering system that maintains sub‐lethal levels of free calcium. If the calcium buffering capacity is exceeded mitochondrial dysfunction can result in excessive ROS production, initiating cell death signaling pathways, and catastrophic energy failure. In this study, we quantitatively characterized the mitochondrial calcium buffering power under a variety of experimental conditions and two calcium loading protocols. This was done with isolated cardiac mitochondria from guinea pigs using calcium‐sensitive molecular dyes. The mitochondrial calcium buffering power was quantified by monitoring the calcium flux from both outside and inside the mitochondria. Here, we report the changes in calcium buffering power caused by oxidative stress and under conditions that promote calcium uptake and retention. Furthermore, we demonstrate that the buffering power is also affected by the way calcium is loaded into the mitochondria. These data provide further insights on the mitochondrial calcium loading capacity and raises the question as to what are the components that regulate free calcium in the system.Support or Funding InformationThis research is supported by the National Heart, Lung, and Blood Institute (R00‐HL121160).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
EHD4 Regulates Prostaglandin E2 Synthesis in Renal Collecting Duct Principal Cells: Potential Implications for AQP2
Authors: Boesen Erika I. , Rahman Shamma S. , Band Hamid 
Published: June 2021

Eps15 homology domain‐containing protein 4 (EHD4) regulates endocytic recycling. We previously reported that global EHD4 knockout (EHD4‐KO) mice have a higher urine flow and lower urine osmolality than wild‐type (WT) mice. Further, the diuretic phenotype in EHD4‐KO mice is associated with a reduced accumulation of aquaporin 2 (AQP2) in the apical membrane of principal cells of EHD4‐KO mice. However, the mechanism by which EHD4 regulates AQP2 localization is not known. EHD4 and AQP2 share common protein interacting partners, therefore we hypothesized that EHD4 interacts with AQP2, thereby regulating its trafficking. To test this hypothesis, we transfected mouse cortical collecting duct principal (mpkCCD) cells with a GFP‐tagged EHD4 plasmid and visualized the localization of EHD4 and AQP2. EHD4 was found to co‐localize with AQP2 in mpkCCD cells. To assess the interaction between EHD4 and AQP2, co‐immunoprecipitation of AQP2 was performed and the presence of EHD4 in the immunecomplex was measured by Western blotting. EHD4 was not detected in the immune‐complex containing AQP2, suggesting that these two proteins may not interact directly. Pointing to a potential indirect mechanism of regulation, evidence from the literature suggests that the paralog EHD1 binds to cytosolic phospholipase A2α, a key enzyme for eicosanoid synthesis. Prostaglandin E2 (PGE2) is a known negative regulator of AQP2 trafficking. Therefore, we hypothesize that EHD4 deletion increases PGE2 synthesis in principal cells, thereby contributing to the reduced membrane accumulation of AQP2. To test our hypothesis we examined urinary PGE2 excretion by female mice and found it to be significantly higher in EHD4‐KO mice (3595 ± 581 pg/day, n = 6) compared to WT mice (1042 ± 131 pg/day, n = 4; P = 0.008). To exclude a role for flow‐mediated stimulation of PGE2 synthesis, we examined the role of EHD4 in PGE2 synthesis in the mpkCCD cell line. PGE2 concentrations in the surrounding media of EHD4‐siRNA treated mpkCCD cells was significantly higher than of scrambled siRNA‐treated cells (84 ± 7 versus 53 ± 5 pg/ml respectively; n = 6; P = 0.0002). Together, these data suggest that EHD4 regulates collecting duct PGE2 production, providing a possible mechanism by which EHD4 regulates AQP2 localization and urine excretion in vivo.Support or Funding InformationAmerican Heart Association Grant‐In‐Aid 17GRNT33661008This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.