bluuebunny/crossref_metadata_2025_split huggingface dataset:
mTOR regulation of trophoblast transcriptome and mitochondrial respiration (1159.1)
Authors: Rosario Fredrick , Cox Laura , Lange Kenneth , Muralimanoharan Subha , Myatt Leslie , Powell Theresa , Jansson Thomas 
Published: June 2021

The role of mechanistic target of rapamycin complex 1 (mTORC1) and mTORC2 in regulating the function of the syncytiotrophoblast, the placental transporting epithelium, is largely unknown. We hypothesized that silencing of mTORC1 in primary human trophoblast (PHT) cells (a) down‐regulates key genes involved in oxidative phosphorylation and ribosome synthesis and (b) inhibits mitochondrial respiration. We silenced raptor (mTORC1 inhibition), rictor (mTORC2 inhibition) or deptor (mTORC1/2 activation) in cultured term PHT cells and performed transcriptome profiling and measured mitochondrial respiration. Inhibition of mTORC1 resulted in a highly coordinated transcriptional response including downregulation of genes in networks related to oxidative phosphorylation, ribosome and lipid biosynthesis whereas inhibition of mTORC2 had no effect on these pathways. Inhibition of mTORC1, but not mTORC2, decreased mitochondrial basal/maximal respiration, ATP coupling, reserve capacity and proton leak, whereas activation of mTORC1/2 had the opposite effect. In conclusion, mTORC1 and 2 differentially regulates the trophoblast transcriptome and mTORC1 is a positive regulator of mitochondrial respiratory function. Placental mTOR signaling is inhibited in fetal growth restriction, which may impair mitochondrial respiration and contribute to placental insufficiency in this pregnancy complication.Grant Funding Source: NIH HD068370
Mcl‐1 and VDAC interaction promotes mitochondrial Ca2+ uptake and ROS production (1159.12)
Authors: Huang Huiya , Li Chi , White Carl 
Published: June 2021

The overexpression of Mcl‐1 protein has been implicated in a wide range of cancer cells and is associated with anti‐apoptotic function and cancer migration and invasion. In the current study the role of Mcl‐1 and voltage‐dependent anion channel (VDAC) interaction in regulating mitochondrial Ca2+ ([Ca2+]mito) and ROS production has been studied. In mouse embryonic fibroblasts (MEF), Mcl‐1 knock‐out caused decreased [Ca2+]mito uptake and mitochondrial ROS production compared to wild‐type cells. Application of two peptides, VDAC‐N‐terminal and L14‐15 shown to disrupt Mcl‐1‐VDAC interaction reduced [Ca2+]mito uptake and ROS levels in the WT and Bax/Bak double knock‐out MEF cells. Similar results were observed in lung cancer cell lines A549 and H1299 which were shown to express high level of Mcl‐1. The presence of N‐terminal and L14‐15 peptides reduced [Ca2+]mito uptake and ROS levels in control cells but were without effect on Mcl‐1 knock‐down cells. In permeabilized A549 cells, higher level of [Ca2+]mito is correlated with a higher ROS level. When given an exogenous [Ca2+] that produced a similar level of [Ca2+]mito, no difference in mitochondrial ROS were observed between control and Mcl‐1 knock‐down cells. Mcl‐1 knock‐down and the presence of the two peptides inhibit cancer cell migration and proliferation as indicated by scratch assay and CyQUANT® Cell Proliferation Assay, respectively, consistent with the role of ROS in regulating migration and proliferation. However, in cancer cell line H460 shown to have low level of Mcl‐1 expression, neither Mcl‐1 knock‐down nor peptides produced a significant effect on mitochondrial ROS level. Taken together, these result suggest that Mcl‐1 and VDAC interaction facilitates [Ca2+]mito uptake, leading to increased ROS production, migration and proliferation in lung cancer cells.Grant Funding Source: American Heart Association
Akt signaling mediates the anabolic action of sympathetic innervation in brown adipose tissue from cold‐exposed rats (1161.6)
Authors: Przygodda Franciele , Buzelle Samyra , Garófalo Maria , Carmo Lilian , Navegantes Luiz Carlos , Kettelhut Isis 
Published: June 2021

Previous studies from our lab have demonstrated that sympathetic nervous system (SNS) exerts an anabolic effect on muscle protein metabolism. However, the physiological role of SNS in the maintenance of brown adipose tissue (BAT) mass, the main site of organism heat production, is unclear. Thus, our goal was to investigate the adrenergic mechanisms controlling BAT protein metabolism. For this, adult Wistar rats were submitted to surgical sympathetic hemidenervation (DEN) of BAT. Six days later, rats were exposed to cold (4oC) for 2 days. The phosphorylation levels of proteins involved in the control of proteolytic pathways and protein synthesis were analyzed by Western blotting. BAT noradrenaline was measured by HPLC. Cold‐exposed animals exhibited an increase in BAT noradrenaline content and in the levels of total (2‐fold) and mitochondrial (60%) proteins. These effects were associated with an up‐regulation in the phosphorylation levels of AktSer (70%), GSK3α (30%) and 4E‐BP1 (2‐fold), which are key mediators involved in protein synthesis. Furthermore, BAT from cold‐exposed animals also showed an increase in the phosphorylation levels of the transcription factor Foxo1, which could inhibit the expression of proteolytic genes. All these effects were abolished in cold‐exposed rats submitted to DEN. In summary, the data suggest that SNS plays an important role in cold‐induced increase in total and mitochondrial proteins in BAT and these effects may be partially mediated by downstream targets of AKT such as FoxO1, GSK3a and 4E‐BP1.Grant Funding Source: Supported by CNPq and FAPESP (13/17111‐0; 12/24524‐6).
ABSTRACT IS WITHDRAWN. (1164.8)
Authors: Kirby Tyler , Fry Christopher , Jackson Janna , Chaillou Thomas , Peterson Charlotte , McCarthy John 
Published: June 2021

We previously reported that hypertrophy of skeletal muscle can occur independent of satellite cell fusion; however, the compensatory mechanism allowing this growth to occur remains unknown. mTOR signaling and total protein content were unaffected by the loss of satellite cell fusion, arguing against a compensatory mechanism involving translation. Alternatively, total RNA content was maintained even though DNA accrual was blocked by the absence of satellite cell fusion. Based on this finding, we postulated that in the absence of satellite cell fusion, myonuclei would increase their transcriptional output in order to maintain RNA levels and thus muscle growth. Preliminary data revealed that RNA:DNA ratio and RNA content per myonucleus was greater in satellite cell‐depleted muscle, which was accompanied by an increase in myonuclear size. Though the significance of this observation remains to be determined, we hypothesize the change in myonuclear shape is indicative of an increase in transcriptional output, specifically ribosomal RNA. Consistent with this idea, we measured under hypertrophic conditions a difference in 28S and 18S content as well as pre‐47S transcript levels in satellite cell‐depleted muscle relative to muscle with a full complement of satellite cells. Thus, it appears myonuclei are able to compensate for the loss of satellite cell fusion during hypertrophy by increasing ribosomal biogenesis.Grant Funding Source: Supported by R01AR060701
Hemodynamic responses to electrical stimulation of carotid sinus in conscious rats (1169.15)
Authors: Castania Jaci , Silva Carlos Alberto , Souza Gean , Dias Daniel , Salgado Helio , Fazan Rubens 
Published: June 2021

Carotid sinus (CS) is an important site of arterial baroreceptors and its electrical stimulation has been used to treat patients with resistant hypertension. This study evaluated hemodynamic responses to electrical stimulation of CS in normal conscious rats. Rats (N=5) had left carotid sinus (CS) carefully isolated, equipped with a pair of electrodes and insulated with a layer of Kwik sil. A catheter was introduced into abdominal aorta throughout laparotomy and exteriorized in the back of the neck, together with the CS electrodes. After two days, conscious rats, under continuous recording of AP and HR, had their CS electrically stimulated, during 20 s, with square pulses of 2 ms, 1 mA and 15, 30, 60 and 90 Hz. Rats showed a basal arterial pressure (AP) and heart rate (HR) of 108±3 mmHg and 353±9 bpm. The recorded voltage of the 1 mA pulses allowed the calculation of carotid sinus resistance that ranged from 4.3 to 7.2 kΩ. A fall in mean AP (Δ=19±2, 28±7, 31±4 and 39±4 mmHg) was observed 5‐9 s after the onset of carotid sinus stimulation with 15, 30, 60 and 90 Hz, respectively. A slight and non‐statistically significant fall in HR (9‐35 bpm) was also observed immediately (1‐3 s) after the onset of CS stimulation. In contrast to electrical stimulation of aortic nerve in conscious rats, currently performed in our lab, CS stimulation affected mainly vascular resistance, once only a slight and non‐significant effect was seem in HR.Grant Funding Source: Supported by CAPES, FAPESP, CNPq.
O‐GlcNAcylation of STIM1 in pulmonary artery endothelial cells (1176.8)
Authors: Vasauskas Audrey , Patel Payal , Patel Neel , Cioffi Donna 
Published: June 2021

Extracellular calcium entry following endoplasmic reticulum store depletion, known as store‐operated calcium entry (SOCE), represents the major Ca2+ signaling event in non‐excitable cells. In the pulmonary endothelium, SOCE through the Ca2+‐selective Isoc leads to increased permeability via inter‐endothelial cell gap formation. Isoc activity may be regulated through post‐translational modification, and indeed, inactivation is phosphorylation‐dependent. Regulation may occur via modification of proteins comprising the channel pore or of accessory proteins such as stromal interacting molecule 1 (STIM1). We showed calcineurin regulates SOCE in pulmonary artery endothelial cells (PAECs) via dephosphorylation of STIM1. In addition to phosphorylation, STIM1 may be regulated by O‐linked β‐N‐acetylglucosamine (O‐GlcNAc), which can have an inverse relationship with phosphorylation on the same residues. Here, we sought to determine whether O‐GlcNAcylation regulates SOC entry in PAECs, and whether this modification has a relationship to phosphorylation. When PAECs were treated with Thaimet G, an inhibitor of O‐GlcNAcylation, SOCE increased. Western blots for O‐GlcNAc showed multiple proteins in PAECs are O‐GlcNAcylated, including a major band at ~85 kDa, likely STIM1. This band decreases upon thapsigargin treatment as phosphorylation increases. These data suggest that O‐GlcNAcylation plays a role and has an inverse relationship with phosphorylation in the regulation of SOCE.Grant Funding Source: Supported by 1F32HL112565‐01, R00HL089361 and R01HL107778
Ephrin‐A1 regulates cell dynamics through molecular activation, mechanical tension, and morphology (1180.13)
Authors: Hur Sung Sik , Yeh Yi‐Ting , Vuong Edward , Min Peter , Nguyen Phu , Wang Kuei‐Chun , Li Yi‐Shuan , Wang Yingxiao , Chien Shu 
Published: June 2021

Ephrins, the Eph family of receptor tyrosine kinases and their ligands, have been shown to be key players in guiding cell migration, polarization, and cell‐matrix and cell‐cell adhesions. Specifically, the ligand Ephrin‐A1 has been shown to regulate cell motility and morphology through the activation of EphA receptors, which signal to PI3K pathway to induce cell contraction. We performed live cell experiments on mouse embryonic fibroblasts (MEFs) to study the interrelations of molecular activation of myosin light‐chain kinase (MLCK) as determined by fluorescence resonance energy transfer (FRET), mechanical tension as measured by using novel 3D intracellular force microscopy (3D IFM), and cell morphology in response to Ephrin‐A1. The results demonstrate positive correlations among the elevation of MLCK, an increase in intracellular mechanical tension, and a reduction of cell area, which indicates an augmentation of contraction. These effects of Ephrin‐A1 were absent in MEFs with PI3K subunit p85b knockout (p85b‐/‐), indicating that p85b in PI3K plays a significant role in mediating the chemical, mechanical, and morphological effects of Ephrine‐A1. This study serves to elucidate the mechano‐chemical regulation of cell dynamics under Ephrine‐A1 guidance.Grant Funding Source: Supported by NIH HL108375 (Shu Chien), NSF: (Shaochen Chen)
TNFα regulates endothelial progenitor cell migration through the NF‐κB pathway (1180.16)
Authors: Prisco Anthony , Hoffmann Brian , Kaczorowski Catherine , Greene Andrew 
Published: June 2021

Many therapies exist to treat myocardial infarctions (MI). Most current therapies focus on management of symptoms rather than regeneration of tissue. Cardiac function has been improved in rodents using bone marrow derived endothelial progenitor cells (EPCs) as a regenerative therapeutic, however human studies have produced inconsistent results as regenerative mechanisms are poorly understood. For an injected EPC to improve cardiac function, the cell must migrate, bind and engraft within the damaged tissue. EPCs are mobilized from the bone marrow to the circulation following MI. One of the inflammatory cytokines released in an MI is TNFα. We hypothesized that an acute dose of TNFα would induce EPC migration toward vessel like structures in vitro. To test this hypothesis, endothelial cells were plated onto a 4 well chamber slide coated with Matrigel substrate to grow vessel‐like structures. After 48 hours, EPCs were treated with TNFα or vehicle and added to the wells containing vessel‐like structures. After tracking the EPCs for 16 hours, a significantly higher fraction of EPCs treated with TNFα than vehicle were found to have migrated towards vessel like structures. Follow up analysis demonstrated that the migratory phenotype was mediated through the NF‐κB pathway, simulated by the TNF Receptor 2. Proteomic analysis suggested several NF‐κB transcriptional targets that were directly tested using siRNA knockdown.Grant Funding Source: NIH Grant HL082798
Holistic multilevel modeling of type‐2 diabetes (1180.5)
Authors: de Graaf Albert , van Ommen Ben 
Published: June 2021

Type‐2 diabetes (T2D), is presenting a rapidly growing health and economic problem. For personalized lifestyle/medical treatment advice, it is essential that computer modelling tools become available to integrate the results from diagnostic tools that probe the function of all involved organs/processes in a single holistic view. Our objective is to build such a model for T2D, an important comorbidity of metabolic syndrome.As a first step towards fully personalized quantitative diagnosis and prediction we developed a descriptive model based on causal loop diagrams at a very high aggregation level that integrates mechanisms across all relevant domains including body weight dynamics, glucose/insulin dynamics, inflammation, gut health, and mental stress. The model integrates qualitative and semi‐quantitative information and expert knowledge.The model was able to simulate the development of T2D over a multi‐year period following different food intake profiles, as well as disease modulation by increased physical activity or mental stress‐relieving lifestyles.In a recently started EU project MISSION‐T2D (www.mission‐t2d.eu), the high aggregation level model will be interlinked with a model at a second aggregation level that describes the integrated dynamics of all processes at the minute‐day timescale. This allows to enrich the model with mechanisms probed by PK‐PD studies and nutritional challenge tests and should pave the way for translating validated multilevel immune‐metabolic models into the clinical setting of T2D.Concluding, we have successfully made a first step towards a multiorgan, multilevel systems health model of T2D that can be used to integrate diagnostic data from different domains for personalized lifestyle/treatment advice.
Cholesterol inhibits ROMK channels by holding PI(4,5)P2 in microvilli (1181.17)
Authors: Liu Bing‐Chen , Yang Li‐Li , Lu Xiao‐Yu , Song Xiang , Li Xue‐Chen , Chen Guangping , Eaton Douglas , Shen Bao‐Zhong , Ma He‐Ping 
Published: June 2021

The kidney plays an important role in maintaining ion balance in the blood. K+ balance is tightly regulated by a K+ channel called ROMK1. We used cultured mouse cortical collecting duct principal cells (mpkCCDc14 line) to determine how ROMK1 is regulated by cholesterol. Previous studies suggest that cholesterol inhibits ROMK channels via a direct mechanism. Surprisingly, our data demonstrate that ROMK1 channels are not located in cholesterol‐rich lipid rafts in cultured CCD cells. Patch‐clamp data demonstrated that a small‐conductance K+ (ROMK1) channel in mpkCCDc14 cells was inhibited by cholesterol and activated by extraction of cholesterol with methyl‐beta‐cyclodextrin (MbetaCD). However, confocal microscopy data showed that ROMK1 was not in the microvilli where cholesterol‐rich lipid rafts are located, but in the planar region of the apical membrane of mpkCCDc14 cells, and that phosphatidylinositol‐4,5‐bisphosphate (PI(4,5)P2), a well‐known activator of ROMK channels, was found only in the microvilli under resting conditions. Interestingly, after cholesterol synthesis was inhibited by lovastatin, PI(4,5)P2 diffused into planar region with a consequence of activation of ROMK channels. Activation of ROMK1 channels by MbetaCD was reversed by addition of exogenous cholesterol, but the activation did not occur when PI(4,5)P2 was sequestered with its antibody. These results suggest that cholesterol inhibits ROMK1 channels, at least in part, by limiting PI(4,5)P2 diffusion from microvilli to planar region of the renal CCD cell apical membrane. Since we have reported that cyclosporine A (CsA), an immunosuppressant drug which causes hyperkalemia, can alter the levels of cholesterol in renal epithelial cells, this study provides a basis for researchers to determine the mechanism of CsA‐induced hyperkalemia.Grant Funding Source: supported by NIH 5R01‐DK067110 to HPM and by NSF of China 81130028 to B‐ZS
Anchored PDE4 controls CFTR conductance in normal and cystic fibrosis airway epithelia (1181.3)
Authors: Richter Wito , Blanchard Elise , Zlock Lorna , Lao Anna , Mika Delphine , Namkung Wan , Xie Moses , Scheitrum Colleen , Gruenert Dieter , Verkman Alan , Finkbeiner Walter , Conti Marco 
Published: June 2021

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that impair its expression or function. PKA phosphorylation is the main mechanism to activate CFTR. However, previous reports indicated that cAMP/PKA activators are ineffective in restoring CFTR conductance in CF epithelia. Our current findings challenge this conclusion. We show that inhibition of type 4 cAMP‐phosphodiesterases (PDE4s) increased CFTR activity in non‐CF primary human bronchial epithelial cells under basal conditions and after β‐adrenergic stimulation, and slowed the return of stimulated CFTR activity to basal levels. In cells homozygous for ∆F508‐CFTR, the most common mutation found in CF patients, PDE4 inhibition alone produced minimal channel activation. However, PDE4 inhibition strongly amplified the effects of CFTR correctors, drugs that increase expression and membrane‐localization of CFTR, and CFTR potentiators, drugs that increase channel gating to reach ~25% of the chloride conductance observed in non‐CF cells. Biochemical studies indicate that PDE4s are anchored to CFTR and mediate local regulation of channel function. Taken together, our results implicate PDE4 as an important determinant of CFTR activity in airway epithelia and support the use of PDE4 inhibitors to potentiate the therapeutic benefits of CFTR correctors and potentiators.Grant Funding Source: Supported by the Cystic Fibrosis Foundation, the NIH, and Cystic Fibrosis Research Inc.
Absence of Slc26a9 results in altered tracheobronchial anion transport and high mortality in neonate mice (1181.7)
Authors: Liu Xuemei , Riederer Brigitte , Anagnostopoulou Pinelopi , Li Taolang , Rausch Brigitte , Duerr Julia , Soleimani Manoocher , Mall Marcus , Seidler Ursula 
Published: June 2021

Background:The anion transporter Slc26a9 is highly expressed in lung and stomach. We observed an excess perinatal mortality in Slc26a9‐/‐ neonate mice. Aim and Methods: In search for the underlying cause, the dead pups were collected, genotyped, and examined histologically. Slc26a9 and CFTR mRNA expression was measured by qPCR in microdissected lung. Slc26a9‐/‐, CFTR‐/‐, and WT neonatal trachea were studied electrophysiologically in Ussing chambers. Results: Dead pups were Slc26a9‐/‐ in >95%. Volume density measurements revealed a strong excess of tracheal and bronchial mucus in the Slc26a9‐/‐ pups. Slc26a9 as well as CFTR mRNA expression was significantly higher in the distal than proximal airways, and significantly higher in neonatal than adult mice. The amiloride‐insensitive, bumetanide‐sensitive short circuit current (Isc) was higher in neonatal Slc26a9‐/‐ than WT trachea, but lower in neonatal CFTR‐/‐ than WT trachea. Surprisingly, forskolin (FSK) + IBMX elicited a significantly higher Isc response (∆Isc) in neonatal Slc26a9‐/‐ than WT trachea. This difference was abolished by preincubation with the CFTR inhibitor CFTRinh172. Conclusions: Neonatal Slc26a9‐deficient trachea displayed a higher electrogenic anion secretory rate than WT, and this difference was abolished by CFTR inhibition. The early death of Slc26a9‐/‐ pups may be related to problems with airway liquid absorption rather than lack of secretion.Grant Funding Source: Supported by DFG Se 13‐4 and 9‐6 to Ursula Seidler
Alkaline pH regulates the epithelial sodium channel (1181.8)
Authors: Ochoa Luis , Peterson Zerubbabel , Collier Daniel , Snyder Peter 
Published: June 2021

The extracellular domain of the epithelial Na+ channel (ENaC) is exposed to highly varied environments, and has been postulated to function as a sensor that modulates channel activity in response to changes in the extracellular environment. In the kidney collecting duct, pH varies widely (4.5‐8.5). We previously found that acidic pH reversibly stimulates ENaC whereas alkaline pH reversibly inhibits ENaC. Eight extracellular domain residues are required for regulation by acidic pH, but mutation of these residues did not alter inhibition by alkaline pH, indicating that alkaline pH regulates ENaC by a different mechanism. Here we tested the effect of extracellular alkaline pH on ENaC current by expressing human ENaC in Xenopus oocytes and recording ENaC Na+ currents as the extracellular pH was altered. We identified the basic residue Arg‐90 in the extracellular domain of γENaC that is in part responsible for ENaC inhibition by alkaline pH. Mutation of this residue to neutral residues dramatically reduced inhibition. When the positive charge was reintroduced by MTSET modification of Cys at this position, the response to alkaline pH was partially restored. Based on homology modeling, we predicted that Arg‐90 is located in close proximity to Glu‐446 in βENaC. Consistent with this prediction, crosslinking of cysteines introduced simultaneously at the two positions (γR90C and βE446C) with MTS‐14‐MTS reduced ENaC current and Na+ self‐inhibition. Together, the data support a role for Arg‐90 in ENaC regulation by alkaline pH. We speculate that titration of this residue induces intersubunit conformational changes that modulate ENaC gating.Grant Funding Source: NIH