bluuebunny/crossref_metadata_2025 huggingface dataset:
The Cardioprotective Potential of Caffeic Acid Phenethyl Ester (CAPE) on H2O2‐induced H9c2 Cell Damage Compared to Common Anti‐oxidants
Authors: Lefkowitz Dylan Samuel , Sandhu Robinder , Kim Alissa , Wieczorek Peter , Castellano Andrew , Barsotti Robert , Young Lindon , Chen Qian
Published: June 2021

Caffeic Acid Phenethyl Ester (CAPE) is a natural compound that has been found to exhibit anti‐proliferative, anti‐inflammative, and anti‐oxidant effects, although these effects have not been fully elucidated on cardiomyocytes. Oxidative stress in the heart contributes to the pathogenesis of various cardiovascular diseases, such as ischemia/reperfusion injury following a myocardial infarction. This study investigated the cardioprotective effects of CAPE against hydrogen peroxide (H2O2)‐induced oxidative injury in rat cardiomyocytes (H9c2) in pre‐treatment (24 hours before H2O2) and co‐treatment (with H2O2), and compared these activities to vitamin C, coenzyme Q10, and caffeic acid. Cell viability was evaluated after co‐treatment and pre‐treatment by a cell counting kit‐8 assay. H2O2 (300 μM to 500 μM) dose dependently decreased cell viability, and 500 μM H2O2‐treated cells (n=4) showed a significantly decreased cell viability of 16 ± 2% compared to control cells (p<0.05). By contrast, co‐treatment (n=5) and pretreatment (n=5) of cells with CAPE (5 to 40 μM) significantly reduced cell damage in a dose dependent manner by 24–29% ± 5–8% (p<0.05). However, co‐treatment and pre‐treatment of caffeic acid (1–40 μM. n=3), vitamin C (100–10,000 μM, n=4), and coenzyme Q10 (0.1–100 μM, n=3) did not show any significant protection on cardiomyocytes against H2O2. These preliminary results suggest CAPE can protect cardiomyocytes against oxidative stress induced by H2O2, which indicate CAPE as a potentially beneficial supplement to maintain cardiovascular health. We plan to investigate the mechanism of action of CAPE in future studies.

Support or Funding Information

This study was supported by the Center for Chronic Disorders of Aging, the Division of Research and the Department of Bio‐Medical Sciences at Philadelphia College of Osteopathic Medicine.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Metformin on the AMPK Pathway, Cystogenesis and Metabolomic Biomarkers in ADPKD Kidney Epithelial Cells
Authors: Li Hui , Naser‐Tavakolian Aurash , Huang Polly , Rivera Daniel , Hallows Kenneth R.
Published: June 2021

Autosomal dominant polycystic kidney disease (ADPKD) is a ciliopathy characterized by the slow and continuous development of cysts derived from renal tubular epithelial cells and is caused by mutations in genes that encode polycystin‐1 (PKD1) and polycystin‐2 (PKD2). Cyst formation and growth results from dysregulated intracellular calcium homeostasis and increased cAMP generation, CFTR activity, and mammalian target of rapamycin (mTOR) pathway signaling. Recent studies in PKD1‐null cells also suggest that there is decreased activity of the metabolic sensor AMP‐activated protein kinase (AMPK) and an excessive reliance on aerobic glycolysis (the ‘Warburg effect’). Here we sought to determine the roles of AMPK and dysregulated metabolism in the pathophysiology of ADPKD, and how the AMPK activator metformin, a potential novel therapeutic for ADPKD, affects these parameters and cyst growth in vitro in both PKD1‐ and PKD2‐null cells. We used immortalized PKD1‐ and PKD2‐null kidney epithelial cells for immunoblotting to measure cell proliferation (mTOR and ERK) and AMPK pathway markers and the levels of key glycolytic enzymes. We also used 3‐D Matrigel cultures to assess cyst growth and Seahorse Extracellular Flux Analyzer assays to evaluate the metabolic phenotype of cells as a function of different treatments. Unlike in PKD1‐null cells, PKD2‐null cells displayed neither excessive activation of the mTOR pathway, inhibition of the AMPK pathway, nor apparent metabolic dysregulation under baseline conditions. Nevertheless, metformin effectively activated AMPK in polarized PKD2‐null cells and significantly inhibited cyst growth in 3‐D cultures, both in the presence and absence of cAMP agonists. PKD1‐null cells had significantly increased cAMP production, with increased expression of the key glycolytic pathway enzymes pyruvate dehydrogenase kinase 1 (PDK1), pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA). Metformin treatment dose‐dependently inhibited expression of these enzymes and ERK activation in PKD1‐null cells. Seahorse metabolic assays showed that PKD1‐null cells have significantly increased basal and compensatory glycolysis. Metformin inhibited oxidative metabolic flux in both PKD1‐ and PKD2‐null cells. We conclude that cystogenesis may occur through differing signaling pathways depending on mutation in ADPKD. Metformin may thus exert beneficial effects in PKD2 mutant cells by mechanisms distinct from those proposed in PKD1 mutant cells and may be in part AMPK‐independent. Additional studies in primary cells and in vivo will be applied to confirm these immortalized cell results.

Support or Funding Information

Supported by NIH‐NIDDK and U.S. Dept. of Defense

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dairy Exosome Effects on Mitochondria Function and Antioxidant Enzymes in Growing Male and Female Rats
Authors: Parry Hailey A. , Mobley C. Brooks , Mumford Petey W. , Romero Matthew A. , Zhang Yufeng , Zempleni Janos , Young Kaelin C. , Roberts Michael D. , Kavazis Andreas N.
Published: June 2021

Exosomes facilitate cell‐to‐cell communication by transporting a variety of molecular cargo among tissues, and have been associated in numerous physiological and pathological conditions. Recent data show that exosomes are not only derived from endogenous synthesis, but can also be obtained from dietary sources (e.g., milk). The aim of this study was to assess the effects of milk exosomes on skeletal muscle and liver mitochondria function, reactive oxygen species (ROS) emission, and antioxidant protein levels in young growing rats. Twenty‐eight day old male (n=12) and female (n=12) Fisher 344 rats were used in this study. For four weeks half of male (n=6) and female (n=6) rats were fed a milk‐based diet containing exosomes (EXO+), and the other animals were fed the same diet which was exosome‐depleted (EXO−). Skeletal muscle and liver mitochondria were isolated for respiration and ROS emission assays. Additionally, protein expression of the antioxidants superoxide dismutase 1/2 (SOD1/2), catalase (CAT), and glutathione peroxidase (GPX) were measured via Western blot in skeletal muscle and liver. State 3 skeletal muscle mitochondria respiration exhibited an interaction effect (p=0.039) with males fed the EXO+ diet having the highest respiration rate. State 4 skeletal muscle mitochondria respiration was higher (p=0.047) in male than female rats independent of diet. State 3 liver mitochondria respiration was not significantly different (p>0.05), but state 4 liver mitochondria respiration was higher (p=0.007) in EXO− rats independent of sex. ROS emission from isolated liver mitochondria was lower (p<0.001) in female rats independent of diet, and liver GPX protein levels were higher (p = 0.005) in female rats independent of diet. No significant differences (p>0.05) were observed in skeletal muscle ROS emission or antioxidant protein levels. In conclusion, the depletion of dairy exosomes from the diet affected mitochondrial physiology in skeletal muscle and liver. In addition, our data show that female rats had lower liver ROS emission and higher antioxidant protein levels (i.e., GPX) compared to male rats. These data may support the mitochondrial free radical theory of ageing since female Fisher 344 rats outlive the male counterparts.

Support or Funding Information

Support provided by AU IGP funds to ANK and MDR. JZ is supported by NIH, NIFA, Gerber Foundation and Egg Nutrition Center, and is a consultant for PureTech.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Long Term Hypoxia Reduces Ca2+ Oscillations in Basilar Arterial Myocytes of Fetal and Adult Sheep
Authors: Reid Casey , Romero Monica , Dobyns Abigail , Wilson Christopher G. , Hessinger David A. , Longo Lawrence D. , Zhang Lubo , Wilson Sean M.
Published: June 2021

Ca2+ oscillations are important in the regulation of many cerebral arterial functions ranging from vasoconstriction to gene transcription. Without these oscillatory waveforms, there are alterations in the signaling pathways regulated by Ca2+. Previous work has shown that long term hypoxia (LTH) due to high altitude exposure can cause cerebral vascular dysfunction including changes in reactivity and morphology in the fetus as well as adult. Studies performed in cerebral arterial preparations have illustrated that LTH in sheep fetus and adult cause whole cell Ca2+ signaling dysfunctions that compromise vascular reactivity and contribute to cerebral arterial pathologies. Based on the premise that Ca2+ oscillations, vasoconstriction, vessel morphology, and LTH mediated cerebral arterial function are interrelated we tested the hypothesis that LTH impairs Ca2+ oscillations. The impact of LTH and maturation on Ca2+ signals in cerebral arterial myocytes was examined using confocal imaging techniques of flou‐4 loaded myocytes of basilar arteries from low (700m) or high altitude (3,801m) near term fetal or adult sheep. LTH decreased the intracellular Ca2+ signals independent of age due to a faster decay in the Ca2+ signal, an effect that could impair vasoconstriction, alter tissue structure, and compromise the regulation of cerebral blood flow. Along with producing smaller Ca2+ events, the data show that LTH inhibits the cell's ability to respond with Ca2+ signals in response to 30 mM potassium‐induced depolarization in fetal as well as adult sheep. LTH and ontogeny may also play interconnected roles in Ca2+ signaling as LTH inhibits distant communication between myocytes in fetal sheep, whereas in adult sheep it stimulates local signaling. These observations illustrate that altitude and maturation each modify Ca2+ signaling behavior in ways that likely impact arterial reactivity and other mechanisms related to the regulation of cerebral blood flow.

Support or Funding Information

This work is supported by The National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development grant number HD083132, by the National Science Foundation under Grant No. MRI 0923559, and the Loma Linda University School of Medicine.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of Thermoregulatory Capability in Weddell Seal Pups
Authors: Pearson Linnea E. , Weitzner Emma , Whoriskey Sophie , Tomanek Lars , Liwanag Heather E.M.
Published: June 2021

One of the greatest metabolic costs for endotherms is the energy allocated to thermoregulation. Weddell seals (Leptonychotes weddellii) are the southernmost breeding mammals, and as such are born into one of the most challenging thermal environments on the planet. During their nursing period, Weddell seal pups must develop the capability to survive both on the ice and in the water. Pups also concurrently shift from the use of fur to a reliance on blubber for insulation. This study examined the thermal and energetic costs associated with development and thermoregulation in Weddell seal pups. Metabolic rate (MR) in air was measured longitudinally for 4 pups every 2 weeks, starting at 1 week of age through the transition to independence (7 weeks); MR in water was measured in the same pups beginning at 3 weeks. Additionally, we assessed the molt status of each pup, and estimated body composition (blubber vs. lean mass) based on an elliptical cones model. There was a high degree of individual variability in mass‐specific MR in air in 1 week old pups (5.96–10.87 ml O2 min−1 kg−1), though in general it remained stable or decreased slightly from 1–7 weeks of age. Mass‐specific MR in water declined from 3 weeks to 7 weeks of age for 2 of the 4 pups, and remained stable for 2 pups. The difference between mass‐specific MR in air and in water also decreased from 3 to 7 weeks for 2 pups and remained steady for 2 pups; but by 5 weeks of age the difference between MR in air and in water was marginal for all 4 pups. Timing of the initiation of the molt and molt duration was also variable; two pups had molted patches by 3 weeks of age, and all but 1 pup were fully molted by 7 weeks of age. All pups gained mass from 1 to 5 weeks of age at an average rate of 2.02 ± 0.09 kg day−1 and the proportion of blubber increased from 1–3 weeks (25% to 38% of body mass). Blubber proportion remained steady from 3–5 weeks (38% to 40% of body mass) because pups gained proportionally more lean mass (61% of total mass gained). By 7 weeks, 3 of the 4 pups had weaned and were losing mass. Timing of MR equivalence in air and water did not vary with molt status (amount of lanugo) or with estimated body composition. Overall, these results indicate that Weddell seal pups have developed their thermal capabilities by ~5 weeks of age to protect themselves against increased heat loss in water, independent of the amount of lanugo fur or body composition.

Support or Funding Information

This project was funded by the National Science Foundation, Office of Polar Programs (#1543539).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Behavioral Thermoregulation of Antarctic Teleosts With and Without Hemoglobin in Response to Acute Thermal Challenge
Authors: Ismailov Iskander I. , Scharping Jordan B. , Andreeva Iraida E. , Friedlander Michael J.
Published: June 2021

Antarctic teleosts C. aceratus (lack hemoglobin, Hb−) and N. coriiceps (possess hemoglobin, Hb+) are extremely stenothermic and live in the Southern Ocean at temperatures near 0°C. While survival of both species is threatened by anthropogenic activities, it is thought to be more so for Hb− fishes which are less tolerant to temperature elevation than Hb+ animals. To examine behavioral thermoregulation of these fishes, freely swimming in a tank, we studied their responses to acute thermal stress. We found that both Hb+ and Hb− species follow generally similar strategy ‐ avoidance‐ and escape‐like transient bursts of locomotion interleaved by periods of relative motionless (possibly, an energy saving maneuver), with some species‐specific differences in onset, duration and intensity. Survival strategy of both species also includes a repertoire of respiratory modularity behaviors, some of which have been previously attributed to parental care in other species of fish. We also found that thermally triggered escape‐like fast S‐starts and C‐turns in N.coriiceps (but not slow C‐turns and withdrawals in C.aceratus) are lateralized. At high temperatures, total reversal (followed by a gradual loss) of lateralization was observed, which may indicate the onset of nervous system malfunction. We conclude that both Hb+ and Hb− Antarctic fishes undergo a variety of ambulatory and respiratory behaviors that appear to represent attempts to avoid and/or escape hazardous temperatures (and, possibly, to adapt to elevated temperatures within a limited range), all similarly sensitive and well‐orchestrated by the nervous system to prevent malfunction, that occurs only when rise in temperature is extreme and unescapable.

Support or Funding Information

Supported by NSF ANT‐1341602.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Body Mass‐Specific Na, K‐ATPase Activity in the Medullary Thick Ascending Limb – Implications for Species‐Dependent Urine Concentrating Mechanisms
Authors: Aw Mun , Armstrong Tamara M. , Nawata Michele , Bodine Sarah N. , Oh Jeeeun J. , Wei Guojun , Evans Kristen K. , Shahidullah Mohammad , Rieg Timo , Pannabecker Thomas
Published: June 2021

In general, the mammalian whole body mass‐specific metabolic rate correlates positively with maximal urine concentration (Umax), irrespective of whether or not the species have adapted to arid or mesic habitat. Accordingly, we hypothesized that the thick ascending limb (TAL) of a rodent with markedly higher whole body mass‐specific metabolism than rat exhibits a substantially higher TAL metabolic rate as estimated by Na, K‐ATPase activity and Na, K‐ATPase α1 gene and protein expression. The kangaroo rat inner stripe of the outer medulla exhibits significantly higher mean Na, K‐ATPase activity (~70%) compared to two rat strains (Sprague‐Dawley and Munich‐Wistar), extending prior studies showing rat activity exceeds rabbit. Furthermore, higher expression of Na, K‐ATPase α1 protein (~4–6‐fold) and mRNA (~13‐fold) and higher TAL mitochondrial volume density (~20%) were seen in the kangaroo rat compared to both rat strains. Rat TAL Na, K‐ATPase α1 protein expression is relatively unaffected by body hydration status or, as shown previously, by dietary Na, arguing against confounding effects from two unavoidably dissimilar diets: grain‐based diet without water (kangaroo rat) or grain‐based diet with water (rat). We conclude that higher TAL Na, K‐ATPase activity contributes to relationships between whole body mass‐specific metabolic rate and high Umax. With the combination of 70% greater Na, K‐ATPase activity and 50% greater total kidney mass:body mass ratio for the kangaroo rat compared to the rat, and assuming all other factors remain equal, the contribution of TAL Na, K‐ATPase activity to overall whole body metabolism would be about 2.5‐fold greater in the kangaroo rat compared to the rat. More vigorous TAL Na, K‐ATPase activity in the kangaroo rat than in the rat may contribute to its steeper Na and urea axial concentration gradients, adding support to a revised model of the urine concentrating mechanism, which hypothesizes a leading role for vigorous active transport of NaCl, rather than countercurrent multiplication, in generating the outer medullary axial osmotic gradient.

Support or Funding Information

NIDDK DK083338 (TP) and DK110621 (TR), NSF IOS‐0952885 (TP), Joint DMS/NIGMS‐NSF DMS‐1263943 (TP), APS/NSF Fellowships (MA).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
AMPK affects thermal tolerance in decapod crustaceans
Authors: Lancor Pierce , Bucicchia Christina , Frederich Markus
Published: June 2021

AMP‐activated protein kinase (AMPK) is a cellular master regulator of energy metabolism, that is activated during stress to ensure a stable ATP pool in the cell. Additional to substantial work on the role of AMPK in mammals, especially humans, it becomes increasingly clear that AMPK is also involved in multiple processes in marine invertebrates, like crustaceans. Following the Oxygen and Capacity‐Limited Thermal Tolerance Hypothesis (OCLTT) marine invertebrates' thermal tolerance is dependent on sufficient oxygen delivery to the tissues and subsequent cellular ATP supply. Recent work has shown AMPK activation during heat stress in the rock crab, Cancer irroratus and the lobster, Homarus americanus. However, heat stress did not activate AMPK in the green crab Carcinus maenas. In the current project we tested whether pharmaceutically activating AMPK prior to a heat stress affects subsequent thermal tolerance. We injected Metformin in C. irroratus and activated AMPK about 3 fold above control levels, without affecting HSP70 protein levels or glucose levels in the hemolymph, indicating that this treatment did not trigger a broader stress response. A subsequent progressive temperature challenge between 11 and 32°C led in the Metformin treated animals to a drop in the maximum heart rate from 150 to 100 BPM at 29 and 23°C (control vs. Metformin treated, respectively), and a drop of the critical temperature, indicated by a switch to anaerobic metabolism and lactate accumulation, from 30 to 26°C. Therefore, AMPK activation diminished thermal tolerance in C. irroratus. Injecting Metformin into C. maenas showed a 30% increase in AMPK activity in the gills, with no substantial increase in AMPK activity in the hepatopancreas or heart. First results on the effect on injecting another AMPK activator, AICAR, don't lead to a substantial increase in AMPK activity yet, and require further adjustments of the treatment protocol.

Pharmaceutically activating AMPK and investigating the subsequent effect on stress tolerance will contribute to a mechanistic understanding of energy allocations during stress in the context of the dynamic energy budget framework.

Support or Funding Information

Supported by a summer research fellowship from the American Physiological Society to P.L., and NSF grant DUE 1431955 to M.F.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enteroglial adenosine A2B receptor signaling contributes to local cytokine production and delays functional recovery following acute inflammation in the mouse colon
Authors: Grubisic Vladimir , Eltzschig Holger K. , Gulbransen Brian D.
Published: June 2021

Glial cells in the enteric nervous system (ENS) contribute to regulation of gut functions by modulating neural reflexes that control motility and secretion. Intestinal inflammation leads to gut dysfunction by inducing neuroplasticity in the ENS. The mechanisms that control local inflammation in the ENS are poorly understood. Resolution coincides with a thousand‐fold increase in adenosine production. Enteric glia express functional adenosine A2B receptors (A2BRs), but the role of glial activation by adenosine is unknown. We hypothesized that enteroglial A2BRs play a role in the progression of the ENS neuroinflammation. We tested our hypothesis by utilizing molecular genetics to ablate A2BRs in glial cells in Sox10::CreERT2+/−/A2BRf/f mice and acute colitis was induced by 2% dextran sodium sulfate (DSS) in drinking water for 7 days. We performed experiments 1 and 3 weeks post DSS treatment to assess the peak and recovery of inflammation. The degree of inflammation was evaluated by body weight change, macroscopic tissue damage, and immunohistochemistry to examine cellular changes in the ENS. Colonic epithelial functions and cytokine/chemokine production were assayed by Ussing chambers and plate array. At peak‐inflammation weight loss, macroscopic tissue damage, and infiltration of CD45+ cells at the level of the myenteric plexus were comparable between the Sox10::CreERT2+/−/A2BRf/f mice and their littermate controls (A2BRf/f). During the recovery, colon wall permeability of DSS‐treated A2BRf/f animals was significantly increased in comparison to their water treated controls, but not in colons from the Sox10::CreERT2+/−/A2BRf/f mice (P = 0.03 and 0.72, respectively, 2‐way ANOVA, water‐to‐DSS comparison). In addition, the ablation of enteroglial A2BRs protected against the DSS‐induced increase in colonic production of cytokines/chemokines at the peak of inflammation (G‐CSF, IL‐1a, IL‐6, IP‐10, MIG), during recovery (IL‐17), or both phases (eotaxin‐1, KC) [P ≤ 0.07 (A2BRf/f) vs P > 0.3 (Sox10::CreERT2+/−/A2BRf/f), 2‐way ANOVA, water‐to‐DSS comparison]. Together, our findings indicate that glial A2BR signaling modulates endogenous immune responses during acute colitis. Glial‐immune interactions mediated downstream of glial A2BR signaling could contribute to gut dysfunction following inflammation. These novel findings could be used in the design of new therapeutics for inflammatory bowel disease.

Support or Funding Information

Dr. Gulbransen's research is currently supported by grants from the Crohn's and Colitis Foundation (CCF; Senior Research Award) and the National Institutes of Health (NIH; RO1DK103723).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Case Study: A Possible Accessory Muscle of the Serratus Posterior Superior Muscle
Authors: Lashley Kerrie Samantha , Granite‐Cohn Guinevere
Published: June 2021

Anatomical variation is defined as the normal range of possibilities in the topography and morphology of body structures. In contrast, congenital anomaly is any structural or functional anatomical finding beyond the normal range of possibilities. This case study describes a congenital anomaly found in a 73‐year old preserved Caucasian male.

This individual presents an atypical left accessory neck muscle originating from the transverse process of the C1 (Atlas) vertebrae. It inserts at more than 90 degrees onto the serratus posterior superior (SPS) proximal attachment at the C7 level.

Head and neck anatomical variations are also present in this individual, which include 4‐headed levator scapulae and 3‐headed splenius muscles bilaterally.

It is thought that this anomaly is a result of embryologic development and it is hypothesize that the atypical neck muscle may reinforce the action of the SPS. Due to the rarity of this finding, a description of such a case is very infrequent in the literature. Identifying anatomical head and neck anomalies are important in understanding pathological and evolutionary implications in our species.

Support or Funding Information

The opinions or assertions contained herein are the private ones of the author/speaker and are not to be construed as official or reflecting the views of the Department of Defense, the Uniformed Services University of the Health Sciences or any other agency of the U.S. Government.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
L‐Arginine Prevents Heme‐Induced Acute Lung Injury (ALI) in a Rat Model of Trauma/Hemorrhage and Resuscitation with Transfusion
Authors: Johnson Fruzsina K. , Durante William , Johnson Robert A. , Rhodes Spencer A. , Stewart Ronald M.
Published: June 2021

Resuscitation with long (> 28 days) stored packed red blood cells (PRBC) promotes acute lung injury (ALI) in our rat model of trauma/hemorrhage. Low nitric oxide (NO) production has been implicated in the development of ALI. Arginase can decrease NO production by decreasing L‐arginine availability for endothelial NO synthase (eNOS). We found that arginase, released from red blood cells (RBC) during prolonged storage, contributes to ALI in our rat model of trauma/hemorrhage. Prolonged storage also increases levels of free hemoglobin (Hb). Heme, a breakdown product of Hb, is metabolized by heme oxygenase to form carbon monoxide (CO). We were the first to report that heme‐derived CO inhibits endothelial nitric oxide (NO) synthase and promotes vasoconstriction in a manner that can be prevented by L‐arginine administration. This study tests the hypothesis that the hemolysis product, heme, promotes ALI in our rat model of trauma/hemorrhage and resuscitation with transfusion in a manner that is exacerbated by limited L‐arginine availability. Fresh PRBC were prepared from rat whole blood. Male Lewis rats were implanted with femoral arterial and venous catheters (trauma), subjected to a 30% hemorrhage and resuscitated with crystalloids and fresh PRBC containing heme alone or with L‐arginine or L‐lysine (competitive inhibitor of L‐arginine uptake). ALI was assessed by pulmonary protein leak (Evans blue dye ‐ EBD) and cytokine‐induced neutrophil chemoattractant‐1 (CINC‐1; rat homologue of human interleukin (IL)‐8) accumulation in bronchoalveolar lavage fluid (BALF). Heme alone caused a 5.6‐fold increase in pulmonary protein leak and 3.5‐fold increase in CINC‐1 accumulation. Co‐administration of L‐arginine decreased heme‐induced pulmonary protein leak by 77% and CINC‐1 accumulation by 32.4%. In contrast, L‐lysine, a competitive inhibitor of L‐arginine uptake, increased heme‐induced pulmonary protein leak by 102% and CINC‐1 accumulation by 106.4%. These data show that the Hb breakdown product, heme, promotes ALI in our rat model of trauma/hemorrhage and resuscitation with transfusion. L‐Arginine co‐administration protects against heme‐induced ALI, while blocking L‐arginine transport by L‐lysine exacerbates heme‐induced ALI. These results suggest that the other hemolysis product, heme, also contributes to ALI in a manner that is greatly exacerbated by decreased L‐arginine bioavailability. We conclude that hemolysis products arginase and heme work in concert to promote ALI during trauma/hemorrhage and resuscitation with transfusion.

Support or Funding Information

Supported by grants AHA 0865241F (FKJ), NIH HL074966 (WD) and USAMRC DOD W81XWH‐07‐1‐0717 (RAJ, RMS).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibitors Targeting IGF‐1r and mTOR Induce Synergistic Cell Growth Inhibition and Enhance Apoptotic Activity in Cancer Cell Lines
Authors: Warshamana‐Greene Gnana (Saku) , Panthi Asmita
Published: June 2021

Insulin‐like growth factor I (IGF‐1) and its cognate receptor (IGF‐1r) show prominent molecular irregularities in many tumor types. Autocrine function for the growth factor is documented in multiple cancers and cell lines. In experimental settings the cell lines show a spectrum of dependency on IGF‐1. Presence of IGF‐1 inhibits programmed cell death with its activation of Akt through the PI3‐Akt‐mTOR signaling pathway. The inhibition extends to chemotherapy‐induced cell death as reported in instances where mammalian target of Rapamycin (mTOR) inhibitors are used pharmacologically for induction of apoptosis. We investigated the effects of concurrent targeting of IGF‐1r and mTOR with potent small molecule inhibitors in cancer cell lines that are dependent on IGF‐1 for cell growth. We show that the drug combination synergistically inhibited cell growth and induced apoptosis at inhibitor concentrations that are minimally toxic to normal lung fibroblast cells but potently inhibits PI3‐Akt activation.

Support or Funding Information

USDA/NIFA # SCX‐311‐17‐14 (1890 research program @SCSU)

NIGMS/NIH IDeA SC INBRE P20 RR‐016461

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Intercellular junctions and endothelial permeability are regulated by cell chirality
Authors: Fan Jie , Ray Poulomi , Lu Yao Wei , Kaur Gurleen , Schwarz John J. , Wan Leo Q.
Published: June 2021

Cell chirality is newly discovered as an intrinsic cell property characterized by the bias of cell polarizing along the left‐right axis. We have recently reported that the endothelial phenotype is right biased; however, little is known about its implications in vascular physiology. In this study, we measured chirality and permeability of human umbilical vein endothelial cells by utilizing micropatterning and transwell culture. Even with low‐level PKC activation, we observed a remarkable reversal of endothelial cell chirality, accompanied by a non‐monotonic change in the monolayer permeability. Using a co‐culture model and immunofluorescence‐based image analysis, we found that uniformity in cell chirality was important for endothelial barrier function such that chirality mismatch between adjacent cells resulted in disrupted cell‐cell junctions leading to increased permeability, which peaked when cell chirality was randomized in the endothelial monolayer. Pharmacological and genetic studies allowed us to determine that PKCα and PI3K/AKT mediated the switch of cell chirality, while other known vascular permeability factors such as VEGF, PAF, and histamine regulated permeability independent of cell chirality. Our study has demonstrated for the first time the role of endothelial cell chirality in vascular permeability regulation and provides unique opportunities for drug delivery across tightly connected cellular sheets.

Support or Funding Information

This work was supported by the National Institutes of Health (OD/NICHD DP2HD083961), National Science Foundation (CAREER CMMI‐1254656), American Heart Association (13SDG17230047), March of Dimes (MOD 5‐FY14‐111). Leo Q. Wan is a Pew Scholar in Biomedical Sciences (PEW 00026185), supported by the Pew Charitable Trusts.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Phenolic‐Enriched Raspberry Extracts and Raspberry Ketone (4‐(4‐ hydroxyphenyl)‐2‐butanone) on Obesity Prevention in a Mouse Model of Diet‐Induced Obesity
Authors: Kshatriya Dushyant , Yeomans Bryn L. , Li Xinyi , Guinta Gina , Bello Nicholas T.
Published: June 2021

Introduction

Red raspberries (Rubus idaeus) contain numerous phenolic compounds with documented positive health benefits. One naturally occurring phenolic, raspberry ketone (4‐(4‐ hydroxyphenyl)‐2‐butanone), is considered the principle flavor component of red raspberries. Raspberry ketone, either as a sole or mixed ingredient, has been marketed as a weight loss supplement.

Objective

To investigate the metabolic signature associated with the weight preventative actions of a phenolic‐enriched raspberry extract compared with raspberry ketone in a diet‐induced obese mouse model.

Methods

Male C57BL/6 mice (8 wk old) received a daily oral dose of vehicle (Veh; 50% propylene glycol, 40% water, and 10% DMSO), raspberry extract low (REL; 0.2g/kg), raspberry extract high (REH; 2g/kg) or raspberry ketone (RK; 0.2g/kg). Coincident with daily dosing mice were placed on a high fat diet (45% Fat kcal) for 4 wks. Feeding, hormonal, and metabolic endpoints were assessed.

Results

For body weight gain, there was treatment effect [F (3, 59) = 5.7, p < 0.005] and treatment × time effect [F (81, 1593) = 5.5, p < 0.005]. On days 20–28, the RK had lower body weights (~9%) compared with Veh (p < 0.05 for all). On days 24–28, the REH had lower body weights (~7%) compared with the Veh (p < 0.05 for all). At the end of the 4 weeks, retroperitoneal [F (3, 55) = 4.8, p < 0.005], epidydimal [F (3, 55) = 10.6, p < 0.005], and inguinal [F (3, 55) = 6.8, p < 0.005] fat masses were lower (~25%) in the RK and REH compared with REL and Veh (p < 0.05 for all). There were no treatment differences in cumulative intake nor in meal patterns. Terminal plasma ghrelin levels [F (3, 57) = 3.5, p< 0.05] were lower (~44%) in REL, REH, and RK compared with Veh (p < 0.05), whereas corticosterone levels [F (3, 57) = 4.4, p <0.005] were elevated (~85%) in the REL compared with Veh (p < 0.05). Indirect calorimetry indicated the RER (VCO2/VO2) [F (3, 28) = 7.4, p < 0.01] was lower (~5%) in REL, REH, REH compared with Veh (p < 0.005 for all). Mean blood pressure [F (3, 148) = 3.0, p < 0.05] was increased in the RK (~19%) and REH (~14%) compared with Veh (p < 0.05 for both). In addition, heart rate [F (3, 148) = 4.2, p < 0.05] was increased in the REH (~30%), REL (~34%), and RK (~38%) compared with Veh (p < 0.05 for all).

Conclusion

Raspberry ketone and phenolic‐enriched extracts of red raspberries have potential to prevent diet‐induced weight gain by alterations in metabolism.

Support or Funding Information

NIH R01 AT008933; USDA‐NIFA NJ06280

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Veterans without mTBI are more likely to benefit from bilateral electrical vestibular stimulation
Authors: Brewer Kelly , Wood Scott , De La Cruz Leslie , Sanjana Faria , Schubert Michael , Serrador Jorge
Published: June 2021

Damage to the vestibular system is often an unrecognized outcomeof mild Traumatic Brain Injury (mTBI). Ocular torsion can be used as a methodfor determining proper vestibular function of the otoliths with the applicationof imperceptible stochastic noise (SN) used to enhance the otolith‐ocular reflex. The goal of this work was to determine if Veterans with vestibular dysfunction withand without a history of mTBI would show a benefit from imperceptible SN.

History of TBI was collected in 122 Veterans (86% male, age 53.7 ± 9.9 years). Thirty‐nine Veteransreported history of mTBI and 76 reported no history. Seven Veterans reported moderateTBI and were excluded from study analysis. History of mTBI was classified as areport of head injury immediately followed by loss of consciousness (less than 30 minutes) or any period of confusion or disorientation. Otolith dysfunctionscreening was performed on 102 Veterans and 41 Veterans returned to participatein repeated testing with and without SN. To assess otolith function andimprovement using imperceptible SN, ocular torsion was collected in response tosinusoidal tilt in the roll plane ± 20 degrees at a frequency of 0.03125 Hz (32sec/cycle). Ocular torsion (OT) gain was used to compare otolith function andimprovement over sham trials using SN.

Veterans screened for otolith dysfunction showed similarrates between groups (58% Ctrl vs 57% mTBI). Veterans with no history of mTBIshowed a significant improvement in OCR with SN over those who reported historyof mTBI (mean improvement 21.3±21% Ctrl vs 1.8±19% mTBI, p=0.008). Statistical significance in OCR improvement remainedwhen adjusted for age (p=0.011). While no difference in mean age was observedbetween groups, Veterans with a history of mTBI showed a higher OCR gain withoutSN (0.09 ± 0.04 Ctrl vs 0.12 ± 0.05 mTBI, result not significant).

These data show that while Veterans with a history ofmTBI demonstrate similar rates of vestibular impairment in otolith‐ocularreflexes, they may be less likely to benefit as a group from bilateralimperceptible electrical stimulation. Further investigation to clarify vestibular‐mediatedpathways of improvements using imperceptible SN are needed and evaluation of unilateraland canal deficits in adjusting for the use of SN may be a beneficialmodification for those that report a history of mTBI.

Support or Funding Information

Supportedby DoD grant W81XWH‐14‐2‐0012

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transsynaptic Tracing of Sensory‐Motor Connections in the Gut‐Brain Axis
Authors: Parker Coltan G , Grako Megan , Berish Brooks , Dailey Megan J
Published: June 2021

The neural gut‐brain axis consists of distinct sensory and motor neurons. Sensory neurons innervating the small intestine transmit meal‐related information to the brain, while motor neurons to the small intestine regulate motility and secretion. Previous research has only partially mapped the brain regions directly connected to the gut‐brain axis, and no research has yet investigated sensory‐motor overlap in those brain regions. Here we used transsynaptic viral tracing to identify the brain regions directly connected to the small intestine and quantify the extent of sensory‐motor overlap in those regions. Because transsynaptic tracing has not been done between the brain and small intestine, we needed to first characterize the viral transmission pattern. A motor‐tracing virus (PRV‐152) was injected to the duodenal wall of adult male Sprague‐Dawley (SD) rats (N=4). Viral transmission was allowed for 3, 4, or 5 days. Brains were processed and virus labeled cells were quantified per nucleus. Progressive viral transmission revealed, for the first time, the sequence of brain regions directly connected to the small intestine through the motor gut‐brain axis. Next, we combined PRV‐152 with a sensory tracing virus (H129 #424) in order to identify sensory‐motor overlap. Both viruses were injected to the duodenal wall of adult male SD rats (N=6). Viral transmission was allowed for 5 days. Brains were processed, and single‐ and double‐labeled cells were quantified per nucleus. We found consistent single‐ and double‐labeled populations across brain regions of the gut‐brain axis, suggesting multiple distinct parallel pathways that persist through the axis. This is the first study to reveal the sensory‐motor organization of the gut‐brain axis, and is a step toward phenotyping the neuronal subpopulations involved in gut‐brain axis control. Further work toward generating a phenotypic map of the axis could enable targeted manipulation of specific gut‐brain axis functions.

Support or Funding Information

This work was supported by USDA Hatch (to MJD) ILLU‐538‐926

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Evolution of Underwater Flight in Manta Rays And Their Relatives (Myliobatidae)
Authors: Hall Kayla C , Hundt Peter , Swenson John , Summers Adam , Crow Karen D
Published: June 2021

Batoids (skates and stingrays), exhibit swimming modes that can be characterized on a spectrum from undulation (>1 wave present along the fin) to oscillation (<0.5 wave present along the fin). The latter is a derived mode of locomotion that evolved in a group of charismatic batoids that have invaded pelagic environments (family Myliobatidae: manta rays, cownose rays, eagle rays). Oscillatory swimming is associated with changes in pectoral fin morphology, including laterally elongated pectoral fins (wings), a posterior redistribution of pectoral fin rays, and the evolution of modified anterior pectoral fin domains called cephalic lobes‐ anteriorly extended appendages used exclusively for feeding in the Myliobatidae. Variation in the number of fin rays in batoid pectoral fins has not been characterized in a comparative or phylogenetic context. To better understand how the batoid body plan was modified in association with a shift in swimming and feeding modes, we quantified fin rays that articulate with the three primary cartilages of pectoral fins and cephalic lobes in myliobatids and their relatives. We found no evidence that additional anterior fin rays evolved in association with the origin of cephalic lobes. Rather, we found that variation in the distribution of pectoral fin rays is associated with the evolution of the oscillatory swimming mode. Further, the degree of partitioning between the cephalic lobes and pectoral fins may be correlated to species‐specific ecology. We found two distinct variations in body plan morphology and skeletal arrangement in the cephalic lobe and anterior pectoral fins within the genus Myliobatis, which appear to be associated with ecology and habitat. Overall, this research has implications for morphological evolution associated with invasion of the pelagic environment, and the biomechanics of underwater flight.

Support or Funding Information

Golden West Women Flyfishers (KCH) and the California State University Council on Ocean Affairs, Science and Technology (KCH), National Science Foundation IOS 1656487 (KDC), and Thematic Collections Network TCN 1701665 (APS).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Clinically relevant congenital reductions in cardiomyocyte N‐glycosylation is sufficient to cause premature death from dilated cardiomyopathy
Authors: Bennett Eric S , Deng Wei , Yip Kay‐Pong , Ednie Andrew R
Published: June 2021

Dilated cardiomyopathy (DCM) often leads to heart failure and an early death, with upwards of 70% of DCM considered idiopathic. The responsible mechanisms, including for congenital DCM, often are not known. The sequential process of extracellularly facing enzymatic protein glycosylation is altered in human DCM and in individuals with DCM risk factors. However, it is not known whether such altered glycosylation is pathogenic or subsequent to DCM onset, nor are mechanisms understood. The importance of glycosylation is further highlighted by the observation that a significant percentage of patients with congenital disorders of glycosylation present with cardiac deficits including DCM of unknown etiology. Previously we showed that changes in sialic acid levels (typically, the terminal glycan residue) attached to specific voltage‐gated ion channel isoforms (VGIC) impact cardiac electrical signaling through electrostatic mechanisms, with these same changes in protein sialylation also causing stress‐induced HF, further suggesting a link between altered glycosylation and aberrant heart function. However, without availability and use of appropriate models, evidence that links, mechanistically, altered glycosylation to heart disease remains largely circumstantial. Thus, here, we created a cardiomyocyte‐specific knockout strain of a single glycosyltransferase gene (GTKO) whose expression was shown to be altered in human end‐stage idiopathic dilated cardiomyopathy. The GTKO mice present with altered myocyte VGIC gating, action potential waveforms, Ca2+ handling, Ca2+ spark activity and contractility, that together contribute to DCM deteriorating into HF with 100% dying early. The data demonstrate that reductions in N‐glycosylation achieved through cardiomyocyte‐specific deletion of a single glycosyltransferase impacts whole‐heart and myocyte functions, drawing a direct link between changes in myocyte N‐glycosylation and heart disease. Thus, this specific GTKO strain provides the first model to investigate the relationship between glycosylation and cardiac disease, showing that clinically relevant changes in cardiomyocyte complex N‐glycosylation are sufficient to cause dilated cardiomyopathy that deteriorates into heart failure and early death.

Support or Funding Information

This work was supported in part by grants from the National Science Foundation [IOS‐1146882, IOS‐1660926 and CMMI‐1266331; E.S.B.]; an American Heart Association, Greater Southeast Affiliate Grant‐In‐Aid [14GRNT20450148; E.S.B.] and Postdoctoral Fellowship [15POST25710010; A.R.E.]; and a grant from the National Heart, Lung, Blood Institute [1R01HL102171‐03; E.S.B.].

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mimicking the activation of the ADP‐glucose pyrophosphorylase from Agrobacterium tumefaciens by site directed mutagenesis
Authors: Alghamdi Mashael , Hussien Rania , Patel Hiral , Dobrzynski Emily , Iglesias Alberto , Ballicora Miguel
Published: June 2021

ADP‐glucose pyrophosphorylase is the enzyme that controls the rate limiting step in the biosynthesis of glycogen and starch in bacteria and plants, respectively. In Agrobacterium tumefaciens, this enzyme is allosterically activated by fructose 6‐phosphate and pyruvate, whereas it is inhibited by AMP. In the crystal structure of the enzyme Ser72 interacts with a sulfate hypothesized to occupy the phosphate of the Fru6P allosteric site. To study the role of this residue, we replaced Ser72 by Ala, Cys, and Trp. In addition, we mutated it to Asp, and Glu to mimic the presence of negative charge of the Fru6P. Replacement by Ala did not significantly change the regulatory properties of the enzyme, indicating that the side chain does not play a critical role. However, we observed that S72D, S72E, S72C, and S72W became insensitive to Fru6P. Cys and Trp residues at position 72 remarkably reduced the specific activity of the enzyme (7.4 and 1.7 U/mg, respectively). On the other hand, S72D and S72E were partially activated (22.3 and 18.9 U/mg, respectively, compared to 8.3 U/mg of the WT). This indicates that the introduction of a carboxylate moiety in position 72 hinders the binding of Fru6P, but partially mimics its presence. These mutants were still activated by pyruvate, which indicates that this region is specifically involved in the activation by Fru6P and not by pyruvate. The mutants were not inhibited AMP inhibitor suggesting that the binding site for Fru6P and inhibitor overlaps. Our results suggest that Ser72 is in the Fru6P regulatory site and, consequently, the sulfate found in the crystal structure must be occupying the site of the phosphate moiety.

Support or Funding Information

This work was supported by NSF Grant MCB 1616851

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.