bluuebunny/crossref_metadata_2025_split huggingface dataset:
Spirulina platensis organic extract increases spare respiratory capacity in RAW 264.7 macrophages to exert an anti‐inflammatory effect
Authors: Pham Tho X , Lee Ji‐Young 
Published: June 2021

Recent evidence supports that crosstalk between energy metabolism and inflammatory processes is a critical determinant for macrophage phenotype. Macrophage energy metabolism greatly influences their activation state. For example, M1, pro‐inflammatory macrophages largely depend on glycolysis while M2, anti‐inflammatory macrophages prefer mitochondrial oxidative phosphorylation (OXPHOS) for their energy generation. We previously demonstrated that organic extract of Spirulina platensis (SPE), a blue‐green algae, exerts a potent anti‐inflammatory effect in vitro and ex vivo. Our recent study also suggests that the anti‐inflammatory effect of SPE is at least partly attributable to the induction of an endotoxin tolerance‐like state in macrophages. Interestingly, although SPE repressed LPS‐induced pro‐inflammatory gene expression as potently as endotoxin tolerant (ET) macrophages, there were some differences between SPE‐treated and ET macrophages. The objective of this study was to determine if SPE alters macrophage energy metabolism to exert its anti‐inflammatory effect in addition to its role in endotoxin tolerance. To determine the energy phenotype, we measured oxygen consumption rate (OCR) and extra cellular acidification rate (ECAR) of naïve, SPE‐treated, and ET macrophages by XF Mito Stress tests using a Seahorse XFe24 Analyzer. Treatment of RAW 264.7 macrophages with 25 mg/mL of SPE or endotoxin tolerization with 10 ng/mL of lipopolysaccharide (LPS) for 12 h increased basal ECAR compared to naïve cells with concomitant increases in the expression of interleukin‐1β and tumor necrosis factor‐α, suggesting that both SPE and LPS activated macrophages to a certain degree. The basal OCR values of naïve cells were the highest, followed by SPE‐treated and then ET cells, which indicates that SPE and LPS decreased cell's dependence on OXPHOS. Interestingly, RAW 264.7 macrophages treated with 25 mg/mL of SPE had the highest spare respiratory capacity (SRC), which is a measure of how much ATP may be produced through OXPHOS in response to a sudden increase in energy demand. When naïve, SPE‐pretreated and ET RAW 264.7 macrophages were stimulated with 100 ng/mL of LPS for 12 h, the SRC was significantly reduced in all treatment groups. During LPS‐stimulation, SPE‐pretreated cells may be better off to meet the energy demands of LPS stimulation as SPE pretreatment significantly increased the SRC. In conclusion, we found that SPE‐pretreated macrophage, but not ET macrophages, had increased the SRC, which may contribute to the potent anti‐inflammatory effects of SPE in macrophages.Support or Funding InformationUSDA AFRI (2014‐01870)
Constitutive phosphorylation of STAT3 by the human serotonin 2C receptor
Authors: Curtis Mary E , Fears Letimicia , Ivy Michael , Fentress Hugh 
Published: June 2021

Serotonin (5‐HT) is a modulatory neurotransmitter in the central nervous system that plays a role in many physiological responses by binding to 16 different receptor subtypes. The 5‐HT2C receptor subtype, a 7‐transmembrane spanning G protein‐coupled receptor, is involved in neuronal excitability, spatial learning, mood, executive function, glucose and energy homeostasis, and appetite. This receptor activates signaling pathways downstream of the Gαq/11 protein, as well as other intracellular proteins that activate G protein‐independent pathways. The structurally similar 5‐HT2A receptor can additionally activate the JAK/STAT pathway, and has been shown to do so following chronic treatment with the atypical antipsychotic and 5‐HT2A and 5‐HT2C receptor antagonist, olanzapine. The purpose of the present study to was to investigate the ability of the 5‐HT2C receptor to activate the JAK/STAT pathway. Human Embryonic Kidney (HEK) 293 cells were transfected with the human 5‐HT2C receptor and stable cell lines were generated. Cells were treated with vehicle, serotonin, olanzapine, or the 5‐HT2CR inverse agonist, SB206553. Proteins were separated by SDS‐PAGE and levels of phosphorylated JAK2 and STAT3 were analyzed via immunoblot. Constitutive phosphorylation of STAT3 was observed in cells containing the 5‐HT2C receptor, while STAT3 phosphorylation was not observed in untransfected cells or cells treated with SB206553 or olanzapine. Treatment with 5‐HT increased the levels of phosphorylated STAT3, while JAK2 phosphorylation was unaffected in all treatment groups. This indicates that agonist binding enhances phosphorylation of STAT3 independently of JAK2 activation. This is the first report of the 5‐HT2C receptor activating this pathway and suggests that this receptor may be involved in growth, cell differentiation, the immune response, and transcription of other STAT activated genes. Future studies will examine activation of this pathway in the adult mouse brain and in the presence of other agonists and antagonists.Support or Funding InformationSupported by the National Science Foundation HRD‐1238723 to Hugh. M. Fentress
A Case of Mistaken Identity: Are Reactive Oxygen Species Actually Reactive Sulfide Species?
Authors: Olson Kenneth R , Gao Yan , Huang Evelyn , Arif Maaz , Arora Nitin , Divietro Alexander , Patel Shivali , Deleon Eric R 
Published: June 2021

Stepwise one‐electron reduction of oxygen to water produces reactive oxygen species (ROS), namely peroxide (H2O2), superoxide (O2.−) and the hydroxyl radical (HO.−). These are chemically and biochemically similar to reactive sulfide species (RSS) that are derived from one‐electron oxidation of hydrogen sulfide (H2S) to elemental sulfur, namely persulfide (H2S2), “supersulfide” (S2.−) and the thiyl radical (HS.−). H2S is also reactive and can act as a RSS. Both ROS and RSS are endogenously generated, signal via reactive protein thiols and have common effector targets. Given these similarities, we hypothesized that extant methods for measuring the former would also detect the latter. Here we compared the sensitivity of five methods commonly employed for measuring ROS to both ROS (H2O2 and superoxide as KO2), and to RSS (H2S, H2Sn where n=2–8, H2S2, H2S3 and H2S4. The methods examined were redox‐sensitive green fluorescent protein (roGFP), 2′, 7′‐dihydrodichlorofluorescein (DCF), MitoSox Red, Amplex Red and H2O2 amperometric electrodes. All methods detected RSS and were as or more sensitive to RSS than they were to ROS. roGFP, arguably the “gold standard” for ROS measurement, was over two‐hundred fold more sensitive to H2Sn (n=2–8) than H2O2. Likewise, ROS and RSS produced dose‐dependent increases in 10 ìM DCF fluorescence, with high (1, 3 mM) H2S giving the most rapid increase in DCF fluorescence. H2O2 and H2S were essentially equipotent, whereas KO2 was considerably less efficacious. Lastly, amperometric H2O2 sensors were nearly 30 times more sensitive to H2S than to H2O2 and two or three times more sensitive to H2S2, H2S3 and H2S4 than to H2O2. The responses of roGFP and DCF to RSS were not diminished by hypoxia (Po2 <4 mmHg) which indicates that they did not involve ROS generation from RSS. The estimated daily production of RSS by cysteine catabolism is similar to estimated ROS production from oxygen consumption, but may exceed it if ROS production is overestimated due to RSS interference. These findings suggest that RSS are far more prevalent in intracellular signaling than previously appreciated and that the contribution of ROS may be overestimated. This conclusion is further supported by the fact that both RSS and antioxidant mechanisms have been present since the origin of life nearly 4 billion years ago, long before the rise in environmental oxygen 600 million years ago. Although ROS are assumed to be the most biologically relevant oxidants, our results question this paradigm. We also anticipate our findings will direct attention toward development of novel and clinically relevant anti‐(RSS)‐oxidants.Support or Funding InformationNSF IOS‐144‐6310 (KRO), NSF GRFP DGE‐131‐3583 (ERD)
Effects of Hypoxia on intracellular H2S and Polysulfides: Implications in O2 Sensing
Authors: Deleon Eric R , Huang Evelyn , Gao Yan , Olson Kenneth R 
Published: June 2021

The mechanism(s) by which cells detect hypoxia and transduce this into appropriate homeostatic responses is a contentious issue. Vascular oxygen sensing is generally attributed to hypoxic generation of reactive oxygen species (ROS). However, we recently found that methods commonly employed to measure ROS cannot distinguish between ROS and reactive sulfur species (RSS) and that RSS and ROS have similar effects on intracellular redox status. Our previous studies suggest that metabolism of hydrogen sulfide (H2S) is the O2 sensor, as H2S mimics hypoxia in numerous systems and H2S metabolism is inversely coupled to O2 availability at physiological levels. In the present study we measure intracellular production of H2S and H2Sn using 7‐Azido‐4‐Methylcoumarin (AzMC) and 3,6‐DI(O‐Thiosalicyl) (SSP4), respectively in HEK‐293 cells and bovine pulmonary artery smooth muscle cells (BPASMC) under both normoxic (21% O2) and hypoxic (0.5% O2) conditions over a 48 hr period. In addition, we attempted to augment H2S and H2Sn production via the known intracellular pathways through addition of substrates (to increase production) and inhibitors (to decrease production). We found that hypoxia increased AzMC fluorescence by 50%, but did not affect SSP4 fluorescence. In normoxia, AzMC fluorescence did not change, whereas SSP4 fluorescence increased two‐fold. These results suggest that hypoxia increases intracellular H2S concentration and concomitantly prevents further the increase in H2Sn concentration. Intracellular H2S and H2Sn was not affected by either cysteine or cystine (10 and 100 uM). Both propargylglycine (10 mM), an inhibitor of cystathionine ã‐lyase and aspartate (1 mM) an inhibitor of cysteine aminotransferase reduced hypoxia stimulated AzMC fluorescence in HEK‐293 cells. Aminooxyacetate, an inhibitor of cystathionione â‐synthase, greatly increased AzMC fluorescence in HEK‐293 cells in normoxia and hypoxia. These are the first measurements of oxygen‐dependent changes in intracellular H2S and H2Sn in living cells and they support our hypothesis that RSS are directly coupled to O2 sensing.Support or Funding InformationNSF Grant IOS‐144‐6310 (KRO), NSF GRFP DGE‐131‐3583 (ERD)
DLP1‐dependent mitochondrial fragmentation mediates heat shock‐induced injury in mouse C2C12 skeletal muscle cells
Authors: Yu Tianzheng , Deuster Patricia , Chen Yifan 
Published: June 2021

Mitochondria regulate cell survival and coordinate cell‐wide stress responses by constantly changing their shapes to alterations in energy demands and/or environmental stimuli. In this study, we investigated the role of mitochondrial morphology in heat‐induced injury in mouse C2C12 skeletal muscle cells. Exposure to heat for 4 hours significantly reduced cell viability (96 ± 3 % at 37 °C vs. 62 ± 6 % at 43 °C, p < 0.01). Mitochondrial morphology analysis revealed that heat exposure caused rapid mitochondrial fragmentation (cells contained fragmented mitochondria: 9 ± 1 %, 47 ± 4 % and 91 ± 2 % at 0, 15 and 30 min, respectively). To identify the molecular mechanism(s) underlying heat‐induced mitochondrial fragmentation, we performed immunoblotting and immunostaining for mitochondrial fission and fusion proteins. Heat exposure increased mitochondrial translocation of fission factor dynamin‐like protein 1 (DLP1). Furthermore, loss of mitochondrial membrane potential and subsequent cleavage of mitochondrial fusion protein optic atrophy 1 (OPA1) in C2C12 cells were noted following heat exposure. Pretreatment with DLP1 chemical inhibitor Mdivi‐1 or knockdown of DLP1 gene expression reduced mitochondrial fragmentation, prevented loss of mitochondrial membrane potential, and improved viability of C2C12 cells in response to heat shock. These results suggest that heat stress is associated with altered mitochondrial dynamics and DLP1‐mediated mitochondrial fission serves a role in heat‐induced cell injury.Support or Funding InformationSupported by Congressionally Directed Medical Research Programs W81XWH‐14‐2‐0133
Prior exercise and changes in plasma volume cause differential baroreflex responses to head‐up tilt
Authors: Charkoudian Nisha , Kenefick Robert W. , Caruso Elizabeth M , Luippold Adam J. , Heavens Kristen R. , Cheuvront Samuel N. 
Published: June 2021

Prior exercise and loss of plasma volume each independently cause cardiovascular changes that can result in hypotension and orthostatic intolerance (OI). However, the profile of volume regulatory factors differs substantially between these conditions. We hypothesized that during isotonic hypovolemia (diuretic‐induced, 3.01±0.23 % body weight loss; n = 6, DIUR), sympathetic neural responses to tilt would be greater than in euhydrated, normothermic subjects (n = 6, EX) who had previously exercised in dry heat (40°C). We measured heart rate (HR), blood pressure (BP) and muscle sympathetic nerve activity (MSNA) during supine rest and head‐up tilt (HUT) to 30 and 45°. Plasma volume decreased 10.2±4.4% in DIUR, and 3.4±3.6% in EX (P<0.01). Supine HR was higher in EX compared to DIUR (82±12 vs. 64±9 beats/min, P < 0.01). However, supine MSNA was not different between groups, either as bursts/min (EX: 24±9 vs. DIUR: 24±10, P>0.05) or bursts/100hb (EX: 30±11 vs. DIUR: 40±19, P>0.05). In contrast to our hypothesis, MSNA responses to tilt were smaller in the DIUR group, both at 30° (EX: 12±7 vs. DIUR 3±4 bursts/100hb, P < 0.02) and at 45°HUT (deltas: EX: 14±8 vs. 3±3 bursts/100hb, P < 0.02). HR responses to tilt were larger in the DIUR group (P < 0.05). Systolic BP tended to be lower in DIUR during tilt (P = 0.05), but diastolic and mean BP values were not different between groups. These data provide an interesting example of differential responsiveness of two arms of the baroreflex (HR vs. MSNA) to successfully maintain BP during tilt in healthy humans based on pre‐tilt differences in cardiovascular and fluid volume status.Support or Funding InformationFunded by USAMRMC; author views not official US Army or DOD policy.
Molecular phenotypes of gills from resident marine versus landlocked freshwater three‐spined sticklebacks (Gasterosteus aculeatus)
Authors: Li Johnathon , Paguio Darlene May , Kültz Dietmar 
Published: June 2021

To analyze the influence of marine versus freshwater (FW) habitats on branchial proteomes of three‐spined sticklebacks, we compared a resident marine (Bodega Harbor, CA) with a landlocked FW (Lake Solano, CA) population. The marine population represents a classical fully plated morphotype whereas the FW population represents a typical low‐plated morphotype as assessed after staining fish with Alizarin Red and analysis of morphometric parameters that define stickleback morphotypes. Unbiased label‐free proteomics was used to identify and quantify over 1700 branchial proteins. Thirty four proteins identified to differ significantly (p<0.05) and by more than 2‐fold in abundance were targeted for data‐independent acquisition and quantitative tandem mass spectrometry (MS2) reaction monitoring with Skyline. Proteins confirmed to be significantly different in abundance were then further analyzed for gene ontology enrichment to assess population‐specific gill functions. Ion transport, oxidative metabolism and redox regulation were significantly enriched in gills of marine sticklebacks while cell/epithelial morphogenesis and proteolysis were significantly enriched in FW sticklebacks. We conclude that these branchial functions and the proteins that mechanistically support them are strong targets of selection when marine sticklebacks invade FW habitats.Support or Funding InformationNational Science Foundation IOS‐1355098.
The Effect of Intracellular pH on Myocardial Calcium Sensitivity in the Anoxia‐Tolerant Painted Turtle
Authors: Fanter Cornelia E , Warren Daniel E , Campbell Kenneth S 
Published: June 2021

The anoxia‐tolerant western painted turtle, Chrysemys picta belli, has the ability to survive without oxygen for over 170 days at 3°C due to extreme metabolic responses that minimize energy usage. Under such conditions, the heart minimizes rate and contractility, resulting in a decreased cardiac output. The onset of anoxia is accompanied by a substantial decrease in intracellular pH, which suggests that the reduction in contractility results mostly from mechanical changes in the myofilament sensitivity to Ca2+ associated with intracellular H+ accumulation. To test this hypothesis, the cellular mechanical properties of turtle myocardium were studied at different pH's from turtles exposed to either 24 hours of anoxic or normoxic conditions at 20°C. Multicellular preparations of turtle ventricles were chemically permeabilized and subsequently attached to a motor and force transducer. Preparations were exposed to an array of pCa solutions varying in pH from 7.4 to 6.6 at 22 °C. The concentration of calcium required to trigger 50% of maximum tension (pCa50) decreased stepwise: pCa50 was 6.2, 5.9 and 5.6 for pH's 7.4, 7.0 and 6.6, respectively. The shift in pCa50 indicates that as pH decreases, 50% tension production requires higher calcium levels. Additionally, maximum contractile force decreased as pH decreased. These data indicate that an increased concentration of H+ results in reduced myofilament sensitivity to calcium and, therefore, a decreased ability for force development. Comparisons between normoxic and anoxic tissues indicated no mechanical differences, suggesting that the metabolic changes seen at the onset of anoxia are likely to be a response of intracellular compositional changes of the chemical environment, rather than structural changes to the contractile apparatus. Overall, pCa50 the painted turtle myocardium is intrinsically more sensitive to calcium concentrations (5.9 at pH 7.0) when compared to human and rat (5.67 and 5.2 at pH 7.0), but is similar to other ectotherms.Support or Funding InformationThis work was supported by a CAREER award from the National Science Foundation.
Glyceroporin HC‐9 in the Freeze Tolerance Mechanism of Cope's Gray Treefrogs
Authors: Stogsdill Brian , Frisbie James , Tsou Pon Ti , Goldstein David 
Published: June 2021

Cope's gray treefrogs Hyla chrysoscelis accumulate glycerol in response to cold as part of their freeze tolerance mechanism. This glycerol may derive from glucose stores in the liver that are broken down and released through glycerol transporters. We therefore hypothesized that these treefrogs would express aquaporin 9 (AQP9), a transmembrane protein from the aquaglyceroporin family that facilitates glycerol transport, predominately in the liver. We also hypothesized that this protein would be up‐regulated during cold acclimation to promote glycerol permeability. To test this hypothesis, we sequenced the AQP9 homolog HC‐9, analyzed RNA and protein expression, and characterized water and glycerol permeability through a Xenopus oocyte expression system.HC‐9 has 315 amino acids which include 2 conserved NPA motifs characteristic of aquaporins, 5 conserved residues typical of glyceroporins, and high sequence homology to human AQP9 (69%) and to AQP9 from Hyla japonica (97%). The mRNA strand also possesses a long untranslated tail that appears to be unique to amphibians and necessary for translation. Characterization using the Xenopus oocyte expression system indicates that HC‐9 is permeable to glycerol but has a poor water permeability compared with human AQP1.In warm‐acclimated treefrogs, HC‐9 mRNA (RT‐PCR) and protein (Western blotting) were expressed in many tissues, with highest levels in liver, stomach, ventral skin, and muscle. Across most tissues, realtime mRNA expression (qRT‐PCR) decreased in cold‐acclimated (5°C) and frozen (−2.5°C) treefrogs, with return to levels near those in warm acclimated animals after return to 5°C for 24 hours. In contrast, protein expression increased in the liver and ventral skin of cold and frozen treefrogs, but remained unchanged or decreased in all other tissues.HC‐9 expression is clearly regulated as a part of the Cope's gray treefrog freeze tolerance mechanism. Contrary to our hypothesis, most tissues have decreased expression of HC‐9 in the cold, which may reduce glycerol permeability to protect cells during the freezing process. Liver is a notable exception by increasing protein expression from warm to cold to frozen conditions. Liver appears to play a critical role in the distribution of the cryoprotectant glycerol during the freeze‐thaw cycle, and this study suggests HC‐9 facilitates in this role.Support or Funding InformationSupported by NSF IOS‐1121457
Effects of Hyperosmotic Stress on Cellular Proteome and Phenotype of Tilapia (Oreochromis mossambicus) OmB Cells
Authors: Wiegers Toni Nicole , Kültz Dietmar 
Published: June 2021

Fish that inhabit an environment of fluctuating salinity (e.g. estuaries, intertidal zones) are frequently exposed to osmotic challenges. These euryhaline fish must cope with unpredictable salinity changes, and thus must respond quickly to alterations in environmental osmolality. Some euryhaline fish, e.g. tilapia, also face irregular and longer periods of high salinity, for instance during seasonal droughts in arid areas. Even though teleosts are osmoregulators, hyperosmotic stress leads to a significant increase in plasma osmolality. To prevent the detrimental effects that result from hyperosmotic stress if unaccounted for, cells must compensate for increased plasma osmolality. A previously immortalized Mozambique tilapia (Oreochromis mossambicus) cell line derived from the brain (OmB) was utilized to expose the mechanisms individual cells employ to survive this stress. Cultured cells were exposed to either control isosmotic media (300mOsm/kg), 450mOsm/kg, which is physiologically relevant for fish plasma, or maximum cellular tolerance levels of 700mOsm/kg for 8 days. Hyperosmotic medium was prepared by increasing NaCl concentration. Cell proliferation and time to confluency, as well as passage numbers, were monitored in treatment and control cells. The morphology and general appearance of experimental and control cells were also periodically monitored via phase contrast microscopy. In addition, proteins that are altered in their abundance by hyperosmolality were identified by label‐free quantitative proteomics to explain cellular phenotypes associated with hyperosmotic stress acclimation. Based on our results we conclude that the tilapia proteome is extensively and specifically modulated during hyperosmotic stress to adjust cellular physiology, stress tolerance, and morphology.Support or Funding InformationThis work was supported by National Science Foundation, IOB 1355098.