bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding Association and Kinetic Characterization of DesD Reveals High Substrate Specificity and Cooperative Behavior
Authors: Tran Leslie , Hoffmann Katherine M.
Published: June 2021

Siderophores, small iron transporting molecules, are secreted by bacteria when there is an insufficient amount of iron. These molecules have high affinity for ferric ions and often outcompete the host chelators. Siderophores synthetase are becoming increasingly associated with virulence in pathogenic bacteria as they are able to evade the human immune response system. One type of siderophore biosynthesis is through the Nonribosomal peptide synthetase Independent Siderophore (NIS) pathway. This project is interested in utilizing the NIS synthetase family's unique fold and novel chemistry as a drug target for new antibiotics.

This project is focused on DesD from Streptomyces coelicolor found mostly in soil. DesD is the last enzyme in the Desferrioxamine E siderophore synthetase pathway. This enzyme is a model for the NIS synthetase family, however, while it has been studied before, there are no good results concerning kinetics. Prior literature has reported a broad substrate specificity but the binding has never been quantified. The preliminary data on binding and kinetics collected from Isothermal Titration Calorimetry (ITC) have revealed high substrate specificity via binding constants, and cooperative kinetic turnover. These baseline studies with be used in future work to quantify inhibitor effectiveness.

Support or Funding Information

This work was supported by a grant from the National Science Foundation (MCB ‐ 1716986) to K.M.H, and by California Lutheran University.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
C. elegans Avoids Toxin‐Producing Streptomyces
Authors: Miller Conrad Laura C. , Tran Alan , Tang Angelina , O'Loughlin Colleen T. , Balistreri Anthony , Chang Eric , Villa Doris Coto , Li Joy , Varshney Aruna , Matthews Sarah , Bremmer Martina , VanHoven Miri K.
Published: June 2021

Species of the non‐motile bacterium Streptomyces protect themselves from predation by the bacteriovore Caenorhabditis elegans through the secretion of nematicides. To evade death by these agents, C. elegans has evolved the ability to detect and avoid the bacterium. We characterize fatty acid analogs, including dodecanoic acid, which elicit a fast avoidance response by C. elegans. We also detect dodecanoic acid that has been secreted by Streptomyces by derivatization and detection with high resolution mass spectrometry. By sensing these signals, C. elegans can escape the nematicidal bacteria that would otherwise be a good food source.

Support or Funding Information

Laura Miller Conrad: National Institutes of Health (5SC3GM118199); Angelina Tang and Sarah Matthews: National Science Foundation (HRD‐1302873); Miri VanHoven: National Institutes of Health (NS087544, GM089595), National Science Foundation (1355202).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
P. gingivalis lipopolysaccharide‐induced exacerbation of oxidized LDL‐mediated macrophage foam cell formation is reliant on TRPV4 channels
Authors: Rahaman Shaik O. , Gupta Nabyendu , Goswami Rishov
Published: June 2021

Although P. gingivalis, a predominant causative agent of periodontitis, has been linked to lipidladen macrophage foam cell formation, a critical process in atherogenesis, the mechanism remains elusive. Emerging data support a role for both a biochemical factor, e.g., lipopolysaccharides (LPS), and a mechanical factor, e.g., matrix stiffness, in regulation of macrophage function, vascular elasticity, and atherogenesis. We have obtained evidence that TRPV4, an ion channel in the transient receptor potential vanilloid family, and a known mechanosensor, is the likely mediator of P. gingivalis lipopolysaccharide (PgLPS)‐induced exacerbation of oxidized low‐density lipoprotein (oxLDL)‐mediated macrophage foam cell formation. Specifically, we found that: i) genetic deletion of TRPV4 or antagonism of its function by a chemical inhibitor blocked PgLPS‐induced exacerbation of oxLDL‐induced macrophage foam cell formation; ii) macrophage TRPV4 activity was increased in response to both PgLPS and pathophysiological range matrix stiffness, and iii) TRPV4 deficiency blocked PgLPS‐induced exacerbation of matrix stiffness‐induced macrophage foam cell formation. Mechanistically, we show that TRPV4 regulates PgLPS‐induced oxLDL uptake but not its cell surface binding in macrophages. Altogether, these findings identify a critical role for TRPV4 in regulating PgLPS‐induced exacerbation of macrophage foam cell formation by modulating uptake of oxLDL.

Support or Funding Information

AHA (13SDG17310007), Startup grant from University of Maryland, NIH (1R01EB024556‐01), and NSF (CMMI‐1662776) grants to SOR.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In vivo electrochemical assessment of dopamine release during conditioned withdrawal in heroin dependent rats
Authors: Pultorak Katherine , Schelp Scott , Oleson Erik
Published: June 2021

The centers for disease control and prevention reports that heroin abuse has doubled for Americans aged 18–25. Drugs of abuse, such as heroin, are commonly thought to increase the concentration of dopamine (DA) in the nucleus accumbens core (NAcc), although their effects on phasic DA release events remains to be fully characterized. In contrast, when an addicted subject is withdrawn from a substance such as heroin, there is a clear suppression of phasic DA activity in the NAcc. Heroin withdrawal presents characteristic symptoms, most predominately increased craving for the drug, which contributes to further abuse. While withdrawal symptoms have been shown to be conditioned to previously neutral stimuli through classical conditioning, there is a lack of electrochemical evidence of conditioned withdrawal and its effects on non‐drug related stimuli. Here, we sought to explore how conditioned withdrawal in heroin addicted rats affects DA. We provided rats 23 hour access to IV heroin over 28 days. The 28 days are split into four blocks of increasing doses ranging from 0.06 mg/kg to 6 mg/kg to more accurately simulate the progression of heroin addiction. During the 28 day history, circadian changes in heroin, food, and water intake were altered and overall consumption of heroin increased. We then, investigated how conditioned withdrawal affects cue‐evoked dopamine release in the NAcc using fast scan cyclic voltammetry (FSCV) during conditioned suppression of food maintained responding. Here, animals were first trained to respond for food under an FR1 schedule for 3 consecutive days where food availability was predicted by a cue‐light placed above the lever. This was followed by 3 consecutive days of conditioned withdrawal training where a tone was conditioned to naloxone‐precipitated withdrawal. On test day, animals responded for food under normal conditions, but the withdrawal associated tone was presented concurrently with cue‐light onset during 50% of trials. By simultaneously presenting the withdrawal paired tone with the food predictive cue light we observed a substantial suppression of DA concentration at the cue light relative to non‐paired trials. These data demonstrated that conditioned suppression of food maintained responding induced by conditioned heroin withdrawal is accompanied by a transient suppression in dopamine concentration

Support or Funding Information

Funding for the project was provided by NSF grant IOS‐1557755, NIH grant R03DA038734, Boettcher Young Investigator Award and NARSAD Young Investigator Award to EBO and an institutional UROP to KJP.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Observing Signaling Dynamics at Endogenous Levels Using a New Class of Biosensors FluoSTEP
Authors: Zhang Jason Zhaoxing , Tenner Brian , Huang Bo , Mehta Sohum , Zhang Jin
Published: June 2021

Intracellular signaling is highly spatiotemporally regulated with specific kinetically‐distinct microdomains. Fluorescent biosensors such as FRET‐based kinase activity reporter, second messenger indicators, and GTPase activation biosensors have facilitated the elucidation of the spatiotemporal regulation for various signaling protein networks; however, overexpression of these biosensors could perturb endogenous signaling. This is especially problematic for GTPase activation biosensors as they contain the GTPase within the sensing domain, thus, causing artificially high GTPase signaling activity. To overcome these challenges, we designed a novel class of fluorescent biosensors named Fluorescent Sensors Targeted to Endogenous Proteins (FluoSTEPs) that utilizes a split fluorescent protein approach to target a specific protein and reconstitute a functional sensor. We have shown that this design is generalizable for various FRET–based biosensors. Furthermore, combining this design with CRISPR knock‐in technology, we demonstrated the application of FluoSTEP in studying cAMP and PKA signaling in the native clathrin‐coated pits and Sec61B microdomains. With the capability of reporting endogenous signaling in the native context of a living cell, FluoSTEP will be powerful tools to investigate spatiotemporal regulation of signaling enzymes such as kinases and GTPases.

Support or Funding Information

NIH R01 DK073368 and R35 CA197622 (to JZ), NSF GRFP (to JZZ)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mu Opioid Receptor Agonists with Dopamine D3 Receptor Antagonism: Toward Developing Analgesics with Less Abuse Potential
Authors: Shah Nilay M. , Schmid Cullen L. , Merritt Benjamin , Kennedy Nicole M. , Ross Nicolette C. , Lovell Kimberly M. , Bannister Thomas D. , Bohn Laura M.
Published: June 2021

Opioid analgesics such as morphine are widely used in clinical practice for the management of moderate to severe pain. While they are the gold standard for painkillers, they are also highly addictive: approximately 10% of patients prescribed opioids for pain‐relief develop an opioid‐use disorder. Abuse of prescription opioids and heroin has become an epidemic in the United States with opioid overdose deaths quadrupling since 1999. Opioids induce their physiological effects through the activation of the mu opioid receptor (MOR). Interestingly, co‐administration of dopamine D3 (DRD3) receptor antagonists with opioids attenuates the self‐administration and rewarding effects of the drugs, but has no effect on their ability to relieve pain. This suggests that the development of compounds that both activate MOR and antagonize DRD3 may be a means to maintain the beneficial effects of opioids, while reducing their abuse potential. We have recently developed a library of small molecules with a novel scaffold that are agonists at MOR. Herein we screened our MOR agonists for antagonism at DRD3 in a DRD3/βarrestin enzyme fragment complementation assay. We identified multiple compounds that antagonize DRD3 that are as potent as Sulpiride, a known DRD3 antagonist. Further characterization of these MOR agonists with polypharmacology at DRD3 may allow for the development of opioid analgesics with reduced abuse liabilities.

Support or Funding Information

Funded by NIDA grant R01 DA033073 to LMB Fellowship to NMS: NSF award 1359369. and TDB, and NSF Undergraduate Research

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 Role of Autophagy During Myocardial Ischemia/Reperfusion Injury
Authors: Ibe Al C. , McIntyre Anahi , Kim Alissa , Kim Hannah , Castellano Andrew , Young Lindon , Barsotti Robert , Chen Qian
Published: June 2021

Autophagy is a housekeeping process to degrade protein aggregates, damaged cytoplasmic constituents, and provides adaptation when cells go through a nutrient stress. However, a debate persists on whether autophagy is beneficial to attenuate or promote cell damage under an ischemia/reperfusion (I/R) insult to the heart. This study tested the effects of an autophagy enhancer (e.g. rapamycin and trehalose) on heart function and infarct size after global ischemia (30 min)/reperfusion (45 min) when given prior to ischemia (pretreatment) or at the beginning of reperfusion (posttreatment). We found that rapamycin pretreatment (25 nM, n=5) significantly restored left ventricular developed pressure (LVDP) to 64±5 % of initial baseline compared to control I/R hearts (n=8) which only recovered to 31±10% of initial baseline (p<0.01). Likewise, trehalose pretreatment (5 mM, n=7) and posttreatment (5 mM, n=6) significantly restored final LVDP to 62±5% and 65±4% of initial baseline, respectively compared to control I/R hearts (both p<0.01). Moreover, infarction percentage after I/R is significantly reduced in rapamycin post‐treated hearts (23±4%, p<0.05) and trehalose treated hearts (20±4% for pretreatment and 15±3% for posttreatment, both p<0.01) when compared to control I/R hearts (41±4%). The preliminary data suggest that autophagy enhancement before ischemia or at reperfusion may be beneficial for reducing I/R injury

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.
The Effects of Hypoxia and Inflammation on Claudin‐1 in CaCo‐2 Cells as a Model Intestinal Epithelium.
Authors: Largoza Gabrielle E. , Cole Chelsea E. , Alana Nicholas B. , Carr Nicholas R. , King Jonathan M.
Published: June 2021

Background

The use of hydrocortisone in association with indomethacin is a known risk factor for spontaneous intestinal perforation in extremely low birthweight infants. Hypoxia, inflammation and non‐steroidal anti‐inflammatory drugs independently alter apical junctional complexes of epithelial cells. We are interested in understanding the combined effects of hypoxia, inflammation and non‐steroidal anti‐inflammatory drugs on the apical junctional complex and barrier integrity in a gastrointestinal epithelial cell model.

Methods

Through the use of CaCo‐2 BBe monolayers, an established in vitro model of the intestinal epithelium, we analyzed the effects of the pro‐inflammatory agents (TNF and IFN‐gamma) and anti‐inflammatory agents (indomethacin and hydrocortisone) on junctional protein expression and localization, cell proliferation using Edu labeling, cellular ATP concentration and barrier function.

Results

Protein expression analyzed by Western blotting techniques demonstrated an alteration of claudin‐1, a tight junction sealing protein. The soluble and insoluble fractions of the cells were analyzed in order to determine the location of these proteins. We determined inflammatory conditions produced a higher concentration in the soluble fraction indicating impaired function. Confocal analysis after 24‐hour exposure to hypoxia showed that the combined use of hypoxia and inflammatory agents elicited no significant change in cell proliferation levels. Cellular ATP levels were analyzed to corroborate proliferation studies. Confocal imaging under the same experimental conditions was also conducted in order to probe for claudin‐1 localization within the cells.

Conclusions

There was no observed significant difference in the cell proliferation rate or marked changes in ATP levels in our model. Therefore, we hypothesize that disturbance in barrier function may be related to specific junctional mechanisms. We found claudin‐1 protein expression and localization to be sensitive to both hypoxia and inflammatory cytokines contributing to impaired barrier integrity. These cellular studies suggest claudin‐1 stability may be a potential target in the progression of spontaneous intestinal perforation.

Disclosure: The view(s) expressed herein are those of the author(s) and do not reflect the official policy or position of Brooke Army Medical Center, the U.S. Army Medical Department, the U.S. Army Office of the Surgeon General, the Department of the Air Force and Army, Department of Defense or the U.S. Government.

Support or Funding Information

San Antonio Medical Foundation Grant, Trinity University, US Air Force 59th Medical Wing Clinical Research Wing

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Organophosphate Pesticide Effects on Neuronal Cytoarchitecture and Morphological Abnormalities in 72 hour Zebrafish Embryos
Authors: Watanabe Taylor H. , Fradinger Erica A.
Published: June 2021

Organophosphate pesticides (OP) are a class of acetylcholinesterase (AChE) inhibitors, which have effects on the central and peripheral nervous systems. OP's inhibit AChE through phosphorylation of the active site serine, which leads to a toxic and potentially lethal buildup of the neurotransmitter acetylcholine (ACh) causing excess stimulation of cholinergic neurons. OP's are widely used to suppress the activity and growth of insect pest populations in both agriculture and household settings. The objective of this study was to examine both organismal and cellular morphological changes caused by OP exposure. Specifically, zebrafish (Danio Rerio) embryos were exposed to sub‐lethal concentrations of Chlorpyrifos (CPF) and its bioactive form Chlorpyrifos‐oxon (CPO), and compared to vehicle controls (MeOH). Gross morphological changes were observed at 72 hours via light microscopy and morphological changes to cholinergic neurons were observed via confocal microscopy of wholemount immuno‐labeled embryos using anti‐ChAT. Light microscopy observations of the embryos showed varying degrees of cardiac edema in both CPF and CPO exposed embryos. Additionally, mild kyphosis was observed in CPF and strong kyphosis in CPO exposed embryos. Confocal observations of the trunk spinal region showed embryos exposed to CPF lost dorsal neural tube cell staining and dark pockets, possibly indicating cell death, were observed along the ventral neural tube. For CPO exposed embryos, cells bodies in the neural tube were disorganized. Additionally, axon projections across the neural tube showed differences in branching and spacing in both CPF and CPO exposed embryos. Together our results showed that OP exposure causes changes in neuronal cytoarchitecture and embryo morphology. Understanding the developmental effects of these pesticides is particularly important since developmental changes have significant effects on adult systems and children represent a vulnerable population.

Support or Funding Information

Department of Defense, ARO 66377‐RT‐REP; Keck Foundation Undergraduate Fellowship to T.H.W.; James Irvine Foundation Chair in Biological Sciences to E.A.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.
Cdc48a AAA‐ATPase and its association with ubiquitin‐like SAMP1 and DNA repair in Archaea
Authors: Dantuluri Swathi , Margulies Shae , Hepowit Nathaniel , Allers Thorsten , Maupin‐Furlow Julie
Published: June 2021

Cdc48/p97/VCP is an AAA ATPase, conserved in eukaryotes and archaea, which is demonstrated to convert chemical energy from the hydrolysis of ATP into the mechanical energy, for either unfolding protein substrates or dislodging protein substrates from complexes. The role of Cdc48 in ubiquitin mediated protein degradation has been well reported; however, its role in DNA repair is less understood so far. Evidence in eukaryotes which implicates Cdc48 in DNA repair is based on the association of Cdc48 with partners and substrate proteins, which are known to be involved in DNA damage response. In our study, we found Cdc48A of Haloferax volcanii co‐purifies with RecJ‐domain (RecJ3 and RecJ4) and RNase J1 homologs in an apparent thiol sensitive complex that binds the oxidative stress‐responsive, ubiquitin‐like SAMP1 in the presence of ATP. RecJ domain proteins are DNases reported to be involved in DNA repair pathways in bacteria. Further analysis revealed an Hfx. volcanii Δcdc48A mutant showed significant difference in survival when compared to its wildtype, on subjecting them to DNA damaging agents like phleomycin and UV. An increase (up to 3.4‐fold; p < 0.05) in transcript levels of cdc48a, recj3 and rnj1 was observed after treatment with the DNA damaging agent phleomycin, suggesting the expression of these genes is correlated with DNA repair. Current focus of this study is to decipher the role of Cdc48A in DNA repair by identifying its partner and substrate proteins. In addition, we are determining the substrate modifications required for signaling the Cdc48A mediated repair pathway using classic biochemistry techniques and genetic manipulations.

Support or Funding Information

The work was supported by funds awarded to Dr. Julie A. Maupin‐Furlow through the Bilateral NSF/BIO‐BBSRC program (NSF 1642283), U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences and Biosciences, Physical Biosciences Program (DOE DE‐FG02‐05ER15650) and the National Institutes of Health (NIH R01 GM57498).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein myristoylation links a high fat diet with prostate tumor progression in mice
Authors: Cai Houjian , Kim Sunjin
Published: June 2021

Objective of the study

Exogenous fatty acids provide substrates for energy production and the biogenesis of the cytoplasmic membrane, but they also enhance cellular signaling during cancer cell proliferation. However, it remains controversial whether dietary fatty acids are correlated with tumor progression. Additionally, the molecular mechanisms explaining the effect of dietary fatty acids in promoting prostate cancer progression remains unknown.

Statement of Methods

In this study, prostate regeneration assay and prostate cancer cell xenografts were applied to study if and how a high fat diet promotes tumor progression in vivo. The amount of Acyl‐CoAs were analyzed by LC–MS/MS analysis. Myristoylation of Src kinase was detected by Click chemistry. Additionally, to examine the molecular mechanisms, proteins were fractionated into cytosol and membrane fraction. Expression levels of Src kinase were detected by immunoblotting.

Summary of the results

We demonstrate that increased Src kinase activity is associated with high fat diet accelerated progression of prostate tumors and that Src kinases mediate this pathological process. Moreover, in the in vivo prostate regeneration assay, host SCID mice carrying Src(Y529F) transduced regeneration tissues were fed with a low fat diet or a high fat diet and treated with vehicle or dasatinib. The high fat diet not only accelerated Src induced prostate tumorigenesis in mice, but also compromised the inhibitory effect of the anticancer drug dasatinib on Src kinase oncogenic potential in vivo. We further show that myristoylation of Src kinase is essential to facilitate Src induced and high fat diet accelerated tumor progression. Mechanistically, metabolism of exogenous myristic acid increased the biosynthesis of myristoyl CoA and myristoylated Src, and promoted Src kinase mediated oncogenic signaling in human cells. Of the fatty acids tested, only exogenous myristic acid contributed to increased intracellular myristoyl‐CoA levels.

Conclusion

Our results indicate that targeting Src kinase myristoylation, which is required for Src kinase association at the cellular membrane, blocks dietary fat accelerated tumorigenesis in vivo. Our findings uncover a molecular basis, which the biosynthesis of myristoyl‐CoA elevates the expression of myristoyl‐Src, and accelerates Src mediated prostate tumor progression in a high fat diet.

Support or Funding Information

NIH (R01CA172495) and DOD (W81XWH‐15‐1‐0507)
Targeting N‐myristoyltransferase1 inhibits prostate cancer progression
Authors: Alsaidan Omar Awad , Kim Sungjin , Li Qianjin , Bielawska Alicja , Cai Houjian
Published: June 2021

N‐myristoyltransferase (NMT) facilitates co/post‐translational myristoylation of several oncogenic proteins, regulating their functions in tumor progression. Overexpression of N‐myristoyltransferase (NMT), which has been shown to be upregulated in a variety of cancers, is associated with androgen independent prostate cancer cells. We demonstrate that genetically ablation of NMT1 inhibited proliferation of prostate cancer cells, tumor growth, and suppressed myristoylation profile of prostate cancer cells. Screening a panel of myristoyl‐CoA analogs against purified human NMT1 protein lead to identifying the B13/LCL4 as an inhibitor for NMT1. B13/LCL4 significantly supressed prostate cancer cell proliferation, migration, and invasion by cell cycle arrest, and inhibited the growth of prostate xenograft tumors with minimal pathological effect on major organs in vivo. Structure activity relationship based optimization of B13 led to LCL204, which showed better inhibitory properties twoards NMT1. We demonstrate that targeting protein myristoylation is a potential therapeutic approach to inhibit prostate tumor progression.

Support or Funding Information

This work was supported by NIH (R01CA172495) and DOD (W81XWH‐15‐1‐0507) to H. Cai

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Detrimental Effects of Aging, Ovariectomy and Weight Gain on Left Ventricular Structure and Function: A Potential Preclinical Model of Early Stage HFpEF
Authors: Bustamante Moises , Garate‐Carrillo Alejandra , Loredo Maria , Garcia Ricardo , Carson Nancy , Ito Bruce , Ceballos Guillermo , Omens Jeffrey , Ramirez‐Sanchez Israel , Villarreal Francisco
Published: June 2021

The incidence of heart failure with preserved ejection fraction (HFpEF) continues to increase and little is known about its pathophysiology. Two‐thirds of HFpEF patients are senile women and risk factors include hypertension and metabolic syndrome. Currently, no drugs are effective for HFpEF treatment and the development of animal models would assist in novel therapy evaluation. We implemented a rodent female animal model of aging, estrogen depletion and weight gain to evaluate the role of these factors in altering cardiac structure/function. Female, 18 month old, Fischer F344 rats were allocated into an aging (A) group, aging + ovariectomy (O) and aging + ovariectomy and 10% fructose (OF) in drinking water (n=8–13/group). For comparison purposes a young (3 month old, n=8) cohort of female rats was included. Left ventricular (LV) structure/function was monitored monthly by echocardiography. At 22 months of age, animals were anesthetized and LV function evaluated using a Millar conductance catheter. Histological measures of chamber morphometry, inflammation and collagen density (fibrosis) were also obtained. OF animals significantly increased body weight vs. A and O. While echocardiography did not detect major alterations in cardiac endpoints, intraventricular pressure‐volume loop analysis evidenced significant (p<0.05) decreases in stroke volume (19% O and 27% for OF vs. A), stroke work (27 O and 31% OF vs. A), cardiac output (43 O and 44% OF vs. A) and increases in Tau (30–35% in A, O and OF vs. young) and LV end‐diastolic pressure volume relationship slope (i.e. stiffness) with ovariectomy in the presence of preserved ejection fraction for all groups. Histomorphometry did not detect changes in chamber dimensions. However, histological analysis indicated progressively increasing levels of inflammatory infiltration, perivascular and interstitial fibrosis with ovariectomy and with fructose supplementation in particular, in the subendocardium (see figure). In conclusion, in the setting of aging, ovariectomy (which depletes estrogens), further deteriorates myocardial microstructure which may facilitate the loss of diastolic and global functional indices in the apparent presence of preserved ejection fraction. This model may serve to understand the role that estrogen depletion and weight gain may have in the development of HFpEF.

Support or Funding Information

DoD 150090 to FV

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
History‐Dependence of the Force‐Velocity Relationship in mdm Mouse Muscles
Authors: Piwinski Lindsay , Rice Nicole , Tahir Uzma , Monroy Jenna , Nishikawa Kiisa
Published: June 2021

The force generated by a muscle is inversely related to the velocity of the shortening. Maximum shortening velocity (Vmax) has been predicted from highly controlled, isotonic conditions which are rarely encountered in vivo. During natural movements, muscles undergo stretch‐shortening cycles, which alter power output and predict higher Vmax than traditional isotonic experiments. We investigated the mechanism underlying the differences in the FV relationship. We hypothesized that for a given force, Vmax is greater during work loop experiments because during stretch a muscle can store elastic energy that can be recovered upon shortening. It is possible that the giant elastic protein, titin, functions as a spring in active muscle and therefore contributes to the storage and recovery of elastic energy during work loops but not isotonic contractions. To test these hypotheses, we compared the FV relationships of soleus muscles from wild type and mdm mice with a deletion in the N2A region of titin. Results showed that Vmax is lower under isotonic conditions compared to work loops in both genotypes but the difference was more than 20% greater in wild type muscles. Thus, titin, cross‐bridge kinetics or some combination of both may explain the observed differences. This work contributes to a growing body of evidence of titin's potential role as a spring in active muscle and sheds light on the underlying mechanism of active muscle stiffness.

Support or Funding Information

NSF IOS‐1456150 awarded to J.Monroy, APS UGSRF

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Liposomal dexamethasone attenuates tourniquet‐induced ischemia‐reperfusion injury in mouse hindlimb
Authors: Tu Huiyin , Zhang Dongze , Wadman Michael C. , Li Yu‐Long
Published: June 2021

Tourniquet‐induced ischemia and subsequent release cause serious ischemia‐reperfusion (IR) injury in the skeletal muscle. Here, we observed the effect of liposomal dexamethasone (lipo‐Dex) in tourniquet‐Induced ischemic reperfusion injury (IR) in mouse hindlimb.

Unilateral hindlimb of C57/BL6 mice was subjected to 3 hours of ischemia by placing a rubber band and followed different periods of reperfusion (1 week, 2 weeks, 4 weeks, and 6 weeks). Before tourniquet was released, lipo‐Dex (7 mg/kg, equivalent to amg/kg/day of Dex for 1 week) was administered via tail vein injection. After 3 hours of ischemia and different periods of reperfusion, gastrocnemius muscle showed significantly morphological and functional damages. TNFα and IL‐1β were overexpressed in the gastrocnemius muscle after 1, 2, 4, and 6 weeks of tourniquet‐induced IR. Treatment with Lipo‐Dex inhibited the elevation of TNFα and IL‐1β, improved structure of the muscle sarcolemma, reduced fragmentation of nicotinic acetylcholine receptor clusters, and promoted recovery of gastrocnemius muscle contraction.

The data suggest that tourniquet‐related IR induces serious skeletal muscle inflammation and degradation. Treatment with lipo‐Dex suppresses IR‐induced inflammation and promotes recovery of skeletal muscle morphology and function from tourniquet‐induced IR injury.

Support or Funding Information

USAMRAA grant from Department of Defense, W81XWH‐17‐1‐0037(PI, Yulong Li).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sink or swim: Early dive behavior in Weddell seal (Leptonychotes weddellii) pups
Authors: Weitzner Emma , Pearson Linnea E. , Tomanek Lars , Whoriskey Sophie , Liwanag Heather E.M.
Published: June 2021

Weddell seals (Leptonychotes weddellii) are a model species for studying diving physiology. However, Weddell seal pups are not born with the elite diving capabilities of their adult counterparts and must develop their skills so they can survive into adulthood. Previous research has excluded an examination of pups' initial diving attempts. The aim of this study was to investigate the development of diving capabilities throughout ontogeny, beginning at 1 week of age, to create a more complete trajectory of development. To do this, we examined the dive behavior and blood oxygen stores of Weddell seal pups over the first seven weeks of life. Flipper‐mounted time‐depth recorders (Lotek LAT1800XLFP) were attached to 1‐week‐old Weddell seal pups (n=8) in Erebus Bay, Antarctica, and were recovered when the pups were 7 weeks of age. Time spent in the water, maximum dive depth, dive duration, bottom time, and post‐dive interval were calculated to characterize dive behavior. Blood samples were collected from three of these pups at 1 week, 3 weeks, 5 weeks, and 7 weeks of age. We measured hematocrit, hemoglobin, and red blood cell counts for all blood samples to assess age‐related changes in blood oxygen stores. Time spent in the water and number of dives per day both significantly increased as pups got older (P < 0.0001). The time pups spent in the water per day ranged between 1 min and 21 h. Although the maximum dive depths for pups ranged between 3 m and 14.78 m, the majority of “dives” were less than 2 m. Hematocrit, hemoglobin, and red blood cell counts also increased throughout early development. Correlations between dive behavior and hematology indicate dive behavior, rather than age, is a better predictor of oxygen storage development. Based on these data, we conclude early experience in the water is an important driver of physiological development in dependent Weddell seal pups. New swimmers may prioritize acclimatization to the water and strengthening their swimming muscles over attempting deeper foraging dives.

Support or Funding Information

This project was funded by a grant from the National Science Foundation (#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.
Intrinsic anti‐inflammatory properties of serum in deep‐diving seals
Authors: Hindle Allyson G. , Bagchi Aranya , Batten Annabelle , Levin Milton , Allen Kaitlin N. , Huckstadt Luis A. , Costa Daniel P. , Zapol Waren M. , Buys Emmanuel S.
Published: June 2021

Diving seals are repeatedly exposed to hypoxia‐reoxygenation and other pro‐inflammatory stimuli such as shear stresses in alveoli from lung collapse and re‐expansion. Moreover, they have a known hyperlipidemic status, which is associated with vascular inflammatory stress in humans and mice. To investigate potential protective strategies in free‐living populations of deep‐diving seals, we examined baseline levels of cytokine biomarkers, as well as the inflammatory response of IL6 gene expression in seal blood cells exposed to an endotoxin (LPS, lipopolysaccharide, 1–1000ng/mL). Despite aspects of their basic physiology that predisposes other mammals to inflammatory stress, cytokine production in healthy Weddell seals (adults and pups) and Northern elephant seals (adults only) following LPS incubation was blunted (10–100X increase in plasma IL6), compared to human samples assayed under the same conditions (>1000X increase). These plasma samples, further assayed using a cytokine panel validated for pinnipeds, revealed that many cytokines were below detectable levels in Weddell seals, with only IL6, IL10 and TNFα exhibiting a dose‐response to LPS. Under isolated, in vitro conditions, Weddell seal monocytes maintained the expected, robust response to stimulation with LPS endotoxin. However, when this response was compared under typical cell culture conditions (using media supplemented with FBS) versus media supplemented with an equivalent amount of Weddell seal serum (WSS), IL6 expression from stimulated monocytes exposed to WSS declined significantly (LPS dose*serum‐type 2way repeated‐measures ANOVA across all doses: F1,6= 8.5, p=0.027 for FBS vs WSS), as did cytokines detected in the cell culture media. To further elucidate the potential anti‐inflammatory properties of seal serum, we repeated the experiment comparing FBS to de‐complemented WSS in RAW cells, a mouse macrophage line. These results validated our findings from seal monocytes, demonstrating that mouse cytokine expression (IL6, TNFα, and Mcp1), was consistently reduced in cells cultured with WSS compared to FBS. Finally, we confirmed that these results held in RAW cells exposed to other TLR ligands, including Pam‐3‐Cys, Poly I:C and CpG DNA (TNFα expression in WSS<FBS, p=0.0001 in each experiment). These findings suggest that seal serum innately possesses anti‐inflammatory properties, which may protect these deep divers from naturally occurring inflammatory challenges such as dive‐induced hypoxia‐reoxygenation and lung collapse.

Support or Funding Information

Funded by the National Science Foundation (Office of Polar Programs).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanisms for Oxygen‐Mediation of Body Size
Authors: Harrison Jon Fewell , Campbell Jacob B. , Lundquist Taylor , Callier Viviane , Cogley Theodore , Fox Trevor , Greenlee Kendra J.
Published: June 2021

Hypoxic‐rearing reduces the adult body size of animals, but the mechanisms responsible remain poorly understood. Holometabolous insects are important models for understanding the regulation of body size. Here we focus on how hypoxic rearing reduces final body size of Drosophila melanogaster. Larval D. melanogaster approximately triple their body mass during the last larval instar. We tested the hypothesis that functional hypoxia develops over time within the instar. Later instar animals had higher whole‐body copy numbers of HIFalpha and HIFbeta RNA and greater quantities of HIFalpha protein (Westerns), providing clear evidence that HIF signaling emerges late in the instar. Later‐instar animals also had greater expression of lactate dehydrogenase genes, and higher capacities for production of lactate, though not higher levels of whole‐body lactate, suggesting that functional hypoxia is not so extreme as to trigger anaerobic metabolism. However, CO2 emission rates increased in response to hyperoxia in late but not early‐instar larvae, providing strong evidence of oxygen limitation of metabolism. Hyperoxia reduced, and hypoxia elevated the RNA copy number of prolyl hydroxylase (whole body), a downstream target of HIF‐signaling. In sum, late‐instar normoxic‐reared larvae experience functional hypoxia, and whole‐body HIF‐signaling is increased with hypoxic‐rearing. Next, to understand the link between functional hypoxia and developmental transitions, we tested whether HIF signaling in the prothoracic glands (which produce ecdysone, aka molting hormone) affects development rate or adult size, using the GAL4‐UAS system to reduce or increase HIF signaling in this endocrine gland. Knockdown of HIF signaling in the prothoracic glands strongly delayed pupariation in 10% but not 21% oxygen atmospheres. Thus HIF‐signaling in the prothoracic gland appears to be an important factor in triggering Drosophila pupariation at a smaller body size in hypoxia, but we found no evidence that this is a key pathway for of the control of molting and size in normoxia.

Support or Funding Information

Supported by NSF IOS 1256745.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analgesic effects of intravenous morphine, fentanyl, sufentanil, and ketamine in a novel rat model of extremity trauma
Authors: Xiang Lusha , Ryan Kathy , Klemcke Harold , Hinojosa‐Laborde Carmen
Published: June 2021

The goal of the current study was to establish a rat model of extremity trauma (ET)for prehospital pain research and validate it using different pain behaviors and analgesics applied via intravenous (IV) injection. Male Sprague Dawley rats (11–13 wk) were briefly anesthetized using isoflurane and ET was induced via clamping there trofemoral tissue groups for 30 sec using an L‐shaped hemostat, followed by closed fibula fracture using a 15‐gauge needle. Rats regained consciousness within 5 minutes after ET. Pain responses to thermal hyperalgesia (paw withdrawal latency of injured hindlimb, PWL), mechanical allodynia (paw withdrawal pressure of injured hindlimb, PWP), and weight bearing (WB) were determined prior to and 90 minutes after ET. Morphine (2 mg/kg), fentanyl (10 μg/kg), sufentanil (1 μg/kg), ketamine (5 mg/kg), or vehicle(saline) was then administered via IV injection, followed by PWL, PWP, WB, and TF assessments at 10, 40, 80, and 120 minutes after analgesic injection. Ninety minutes after ET, the PWL, PWP, and WB were significantly decreased by 61 ± 4%, 64 ± 8%, and 65 ± 4%, respectively, compared to baseline values. These pain behaviors were steadily maintained throughout the 3–4 hours of observation. Compared with the saline‐treated group, opioid analgesics significantly increased PWL during more than 80 minutes, with sufentanil exhibiting the highest analgesic effect. An increase in PWL was only observed at 10 minutes after ketamine injection. PWP was transiently increased with opioid analgesics for 10 to 40 minutes but was not changed with ketamine. WB was improved with opioid analgesics for atleast 2 hours but only for up to 80 minutes after ketamine injection. The ET model includes long bone fracture and soft tissue injury but no fixation surgery, mimicking prehospital extremity trauma, and produces acute, steady, and reproducible trauma‐related pain behaviors. Our model is clinically relevant regarding the pain behaviors and established responses to common analgesics.

Support or Funding Information

This study was funded by the US Army Clinical and Rehabilitative Medicine Research Program, Joint Program Committee 8, Applied Pain Research Award.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sex Specific Differences in Cardiac Fibrosis
Authors: Conger Autumn , Milburn Gregory , Blair Cheavar , Guglin Maya , Wells Gretchen , Waikel Rebekah , Campbell Kenneth
Published: June 2021

Cardiovascular disease is the leading causes of death in America, which prompted a concerted effort to better understand the causes and to develop innovative therapies. The etiology of heart disease is complex and depends on several factors such as age, race, and sex, but previous research conducted contains gaps, as it often did not look for a difference between these groups. This study aims to fill one aspect of this research gap by examining sex‐specific differences in cardiac fibrosis in failing and non‐failing human hearts. Cardiac fibrosis is a result of cardiac remodeling and causes decreased heart function post cardiac damage. A previous study, conducted in our lab, used Nanostring analysis to examine sex‐specific differences in the expression of genes related to fibrosis. The results of this study showed certain gene expressions were specific to sex or had an interaction between the sex and heart failure status. Several of these genes were regulators of collagen, a common type of cardiac fibrosis. We continued this study by quantifying collagen in human heart samples, collected from heart transplants and non‐viable organ donors. These samples were stained using an established picrosirius red staining technique, turning the collagen tissue red and normal cardiac tissue yellow. These samples were then quantified using a k‐means cluster, via a program written in the lab, to eliminate any bias in determining red and yellow tissue. The results are pending as there are still samples left to be completed. We hope to continue pursuing this line of inquiry into sex‐specific differences in heart disease by examining passive stiffness, of which collagen is a major component.

Support or Funding Information

Penny Warren Award for Clinical and Translational Research

AHA 15GRNT2546003(Campbell:PI)

NIH UL1 TR000117 (Campbell: Core Leader)

NSF 1538754 (Campbell: Co‐I)

NIH R01 HD090642 (Campbell: PI of sub‐contract)

NIH U01 HL133359 (Campbell: Co‐PI)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Shifting Nitrogen Balance Induces Arousal from Hibernation in Arctic Ground Squirrels
Authors: Rice Sarah A , D'Alessandro Angelo , Barati Zeinab , Gehrke Sarah , Reisz Julie A , Drew Kelly
Published: June 2021

Hibernators, such as arctic ground squirrels (AGS), undergo brief periods of euthermy (termed interbout arousals) where they radically change their physiological and metabolic state. These interbout arousals rebalance metabolic and nitrogen homeostasis. A key metabolic characteristic of a hibernation bout is a progressive increase in glutamine in liver, representing an increase in nitrogen load, which is resolved over the interbout arousal. We hypothesized that increasing the nitrogen load over a bout of torpor would induce arousal. We tested the impact of increased nitrogen load on timing of arousal and metabolic profile by blinded infusion of ammonium acetate at 6 and 30 mg/kg/hr for a 12 hour period early in a torpor bout. Sodium acetate (6 and 30 mg/kg/hr) and saline served as controls. Plasma was sampled at 0, 6, 12, 30 hour from infusion start and at points defined as mid and late torpor. We monitored the core body temperature and rate of oxygen consumption to assess the hibernation state of the animal. The metabolic impact of exogenous nitrogen load was tracked over time in plasma with ultra high‐performance liquid chromatography mass spectrometry (UHPLC‐MS) metabolomics.

Infusion with the highest concentration of ammonium acetate resulted in a shorter bout length than infusion with saline (Table 1). The effect of infusions on metabolic profiles is pending analysis. In conclusion, results support the hypothesis that nitrogen load limits torpor bout length. Further work is needed to determine if shifting nitrogen balance in hibernation mimics endogenous mechanisms which naturally induce arousal.

This work was performed in conformance with the FASEB Statement of Principles for animals used in research.

Support or Funding Information

This work was supported by the NSF award number 1258179 and NIH 20GM103395.

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