bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct Current Electric Fields Induce Collective Migration of Human Corneal Epithelial Monolayers
Authors: Contreras Adam , Zhu Kan , Guo Liang , Lee Rachel , Campanello Leonard , Losert Wolfgang , Zhao Min
Published: June 2021

Bioelectricity is an endogenous biological electric field (EF) that can be measured in vivo. A biological direct current (DC) EF is produced by ion fluctuations, and is a common response to wounds in various tissues. Our lab previously illustrated DC EF responses in corneal wounds in rodent models in vivo and in human corneal epithelial cell (hCEC) culture. We have observed that both endogenous DC EF and applied exogenous DC EF are capable of guiding cell migration in the process of electrotaxis. However, we have yet to examine exogenous DC EF responses in epithelial monolayers, which are more representative of a wound edge. Our aim was to characterize collective migration of epithelial monolayers to model electrotaxis of a wound edge. We hypothesized that exogenous DC EF can improve collective migration in hCEC monolayers, and therefore DC EF might be applicable for medical treatments of corneal wounds. To test the first part of this hypothesis, we applied exogenous DC EF to hCEC monolayers. We used a standard method of cell tracking combined with an advanced method of particle image velocimetry (PIV) analysis to extrapolate collective migration data. We observed that applications of DC EF to hCEC monolayers induced collective migration and directionality ‐ the directional precision of migration parallel to the EF. Increasing DC EF voltages of 0, 50, and 200 mV/mm2 positively correlated with increasing migration rates. In controls (0 mV/mm2), we observed no directionality, but at DC EF voltages of 50 and 200 mV/mm2 we observed a comparable directionality response, guiding cell migration parallel (0 degrees) to the DC EF. Further, directionality was promptly reversed following DC EF reversals (180 degrees). We concluded that DC EF provides a precise guidance signal for hCEC monolayers that might be applicable for corneal wound healing therapies. Future investigations will aim to dissect cellular mechanisms of the collective migration response to DC EF and to examine the feasibility of DC EF applications to corneal wounds in vivo.

Support or Funding Information

NIH, NEI ‐ 2R01EY019101‐05A1 to MZ; DOD, MURI ‐ FA9550‐16‐1‐0052 to WL and MZ

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Neural Circuits and Olfactory Responses to Neurotropic Viruses in Trout
Authors: Mancha Fabiola , Salinas Irene , Huertas Mar
Published: June 2021

Vertebrate olfactory receptors (OR) are directly exposed to microorganisms, such as viruses, due to their direct contact with the external environment. A previous study showed that nasal delivery of rhabdovirus IHNV (Infectious hematopoietic necrosis virus) in fish activate nasal immune responses marked by an increase of chemokine CCL19 and prostaglandin synthase expression in olfactory epithelia (OE), and infiltration of CD8+ cells in the OE.

We hypothesize that nasal immune responses are activated by action potential signals generated by activated olfactory receptor (OR) neurons in the OE and olfactory bulb (OB). Moreover, this neural circuit can be traced from a specific OR cell type (crypt cell) in the nose to a specific site in the olfactory bulb. We tested our hypothesis by measuring olfactory responses to live attenuated IHNV virus by electro‐olfactogram (EOG). We also visualized the IHNV neural circuit after activation of specific OR, and consequent internalization of molecular receptor and IHNV mixed with Alexa dextran 488 3000 MW.

Our results showed different EOG olfactory responses to live attenuated IHNV and to the medium where the virus was grown (negative control) in rainbow trout. Olfactory responses followed a dose‐response pattern typical of OR. Cross adaptation studies also showed that live attenuated IHNV activates a set of receptors different from those activated by virus‐free supernatants. Recordings of the OB responses by electroencephalogram are under development. Preliminary tracings show fluorescent oval shaped OR in the apical border of the olfactory lamella (putative crypt cells) that extended to the ventral side of the olfactory bulb. This neural circuit differs from those visualized after exposure of trout OE to the food odorant serine.

Combined, our results adds evidence for a new olfactory function in trout, which serves as a first layer of pathogen detection in vertebrates.

Support or Funding Information

This material is based upon work supported by the National Science Foundation under Grant No. 1755348

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Human Skeletal Muscle‐on‐a‐Chip
Authors: Naik Akshata R , Pernal Sebastian , Lewis Kenneth T , Wu Yaobin , Wu Hongkai , Carruthers Nicholas J , Stemmer Paul , Jena Bhanu P
Published: June 2021

A comprehensive understanding of the emergent properties of in vitro skeletal muscle growth and development is still in its infancy. To address this gap in our current knowledge, we have developed a stretchable micropatterned 3D human skeletal muscle platform that recapitulates organized and parallel growth of muscle cells and fibers as opposed to the randomly oriented cells growth on a 2D glass surface. Mass spectrometry of the muscle cells growing on the 3D platform express key myogenic proteins for myoblast fusion required in the formation of muscle tissue, and proteins involved in mitochondrial health and biogenesis, in contrast to cells growing on 2D glass surface. These results demonstrate that the engineered human muscle cells grown on the 3D platform holds great promise to further establish the emergent properties of in vitro skeletal muscle growth and development for a wide range of biomedical applications such as transplants, drug screening and gene therapy.

Support or Funding Information

Interdisciplinary Biological Sceinces (IBS) fellowship

Graduate Research Assistantship, Department of Physiology

Richard Barber Fellowship

National Science Foundation grants EB00303, CBET1066661

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Commonly Abused Drugs Differentially Increase the Frequency and Amplitude of Accumbal Transient Dopamine Release Events
Authors: Everett Thomas , Leman Ryan , Lopez‐Beltran Hendrick , Chaman Oniza , Isaacs Dominic , Oleson Erik B
Published: June 2021

The rewarding and reinforcing effects of abused drugs are thought to be mediated by their ability to increase brain dopamine levels. The canon holds that all drugs of abuse increase dopamine release in a brain region called the nucleus accumbens (NAc) but, due to the temporal constraints of previously available neurochemical techniques, it remains unknown how distinct drugs of abuse alter dopamine release in real time. A real time assessment is important due to the nature by which dopamine neurons fire. In awake and behaving rats, dopamine neurons fire in two distinct patterns. At ‘rest,’ dopamine neurons fire in a low‐frequency pacemaker fashion that is thought to produce a tone on high‐affinity dopamine receptors in the NAc. When presented with motivationally relevant stimuli, dopamine neurons fire in high‐frequency bursts. The phasic bursts of dopamine neural activity contribute to the generation of subsecond release events in the NAc that are sufficient in concentration to occupy low‐affinity dopamine receptors. As the activation of these low‐affinity receptors is thought to be particularly important in strengthening action‐outcome associations, we are especially interested in investigating how drugs of abuse alter their patterns of release. Fast‐scan cyclic voltammetry (FSCV) allows for the detection of dopamine release events with a temporal resolution of milliseconds, making it an ideal tool with which to examine how drugs of abuse alter transient dopamine release events in real time. We obtain two primary dependent measures of dopamine release, the frequency at which they occur and the total concentration of each event.

Here, we are using FSCV to compare how different drugs of abuse alter the frequency and amplitude of dopamine release events in the awake and behaving rat. Drugs of investigation include: ethanol, diazepam, zolpidem, WIN55,212‐2, methamphetamine, methylenedioxymethamphetamine (MDMA) and heroin. Preliminary data show that the sedative‐hypnotics ethanol, diazepam and zolpidem all increase the frequency but decrease the amplitude of dopamine release events; although the ethanol amplitude effect is biphasic. Heroin, the synthetic cannabinoid WIN55,212‐2, and the psychostimulant methamphetamine increase both the frequency and amplitude of dopamine release events. The psychostimulant and tryptamine psychedelic MDMA increases the amplitude of dopamine release events but not their frequency. Together, these data reveal that the effects of abused drugs on transient dopamine release events varies across drug classes.

Support or Funding Information

Funding for this work was provided by NSF grant IOS‐1557755, NIH grant R03DA038734, Boettcher Young Investigator Award and NARSAD Young Investigator Award to EBO

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bulk Material Properties of Beta–Bulge Peptide Hydrogels For Tissue Engineering
Authors: Dreher Stephanie Marie , Salick Daphne A. , Haines‐Butterick Lisa , Schneider Joel P.
Published: June 2021

Hydrogels are porous, physically rigid, three dimensional matrices primarily composed of water which show promise as scaffolds to aid in tissue regeneration.1,2 The Schneider laboratory has developed a class of peptides that adopt β‐hairpin structure and self‐assemble into a hydrogel in response to specific stimuli.3,4,5,6 The peptide MAX8 consists of alternating valine and lysine residues on each β‐strand flanking a type II′ β‐turn (‐VDPPT‐) with a glutamic acid at position 15. The kinetics of self‐assembly for the MAX8 under cellular encapsulation conditions are fast enough to result in homogeneous gel‐cell constructs.7

MAX8 must be prepared by chemical synthesis due to the unnatural amino acid DProline in the turn region. A new sequence, BBH8, was designed to contain all natural amino acids which can possibly be overexpressed. BBH8 was first chemically synthesized and its bulk material properties characterized under physiological and cell culture conditions. Data show that BBH8 behaves analogously to MAX8. Preliminary data also demonstrate that chondrocytes can be encapsulated with homogeneous cellular distribution and remain viable in the network for at least 3 hours. This suggests that BBH8 is an excellent candidate for tissue engineering and possible production by fermentation. Funding provided by NSF CHEM0348323, NIH R01 DE016386‐01, and the Howard Hughes Medical Institute.
Characterization of a benzoylformate decarboxylase and a NAD+/NADP+‐dependent benzaldehyde dehydrogenase from Pseudomonas stutzeri ST‐201
Authors: Saehuan Choedchai , Rojanarata Theerasak , Wiyakrutta Suthep , Meevootisom Vithaya , McLeish Michael J.
Published: June 2021

Both D‐phenylglycine aminotransferase and benzoylformate decarboxylase (BFDC) activity were detected in cultures of P. stutzeri ST‐201 following induction with D‐phenylglycine. Conversely, induction with benzoylformate enhanced only BFDC activity. Colony hybridization was used to identify two genomic DNA fragments which, when combined, contained two open reading frames whose gene products were characterized as a benzoylformate decarboxylase and a NAD+/NADP+‐dependent benzaldehyde dehydrogenase. The kinetic properties of the BFDC was similar to those of the BFDC found in the mandelate pathway of P. putida ATCC 12633. However, the Km values for NAD(P)+ of the benzaldehyde dehydrogenase were significantly higher than those of the P. putida enzyme. Based on sequence and structural alignments this was likely to be due to a change from leucine to arginine at position 175. Given that P. stutzeri ST‐201 was unable to grow on either isomer of mandelate, and that sequencing indicated that dpgB did not form part of an operon, it appears that the two enzymes form part of a D‐phenylglycine, rather than mandelate, degrading pathway.

This work is supported in part by the CHED and Faculty of Graduate Studies, Mahidol University, and granted by TRF through the RGJ‐Ph.D. Program No. Ph.D/0023/2548. The work was also supported by NSF EF 0425719.
Exploring the Strength of Subunit Interfaces Using Guanidine Hydrochloride and Dynamic Light Scattering
Authors: Marion Jimmy , Bell Ellis
Published: June 2021

Dynamic light scattering is used to determine changes in mass and mean diameter of particles. Through the combination of varying concentrations of guanidine hydrochloride with either monomeric or multimeric protein samples we have developed a novel approach using Dynamic Light Scattering that has allowed us to test not only the strength of a protein's subunit interface but also to test the stability of its tertiary structure. Model systems, such as monomeric Bovine Serum Albumin (BSA) and tetrameric hemoglobin, with a known dissociation pattern, are used as fundamental examples to this light scattering approach. Watermelon Glyoxasomal Malate Dehydrogenase (dimer), E. coli 3Phosphoglycerate Dehydrogenase (tetramer) and Bovine Glutamate Dehydrogenase (hexamer) are used as complex systems to further investigate the approach. By measuring DLS at different guanidine hydrochloride concentrations in the range 0–2M dissociation of the oligomers can be followed where little change in tertiary structure occurs. By including ligands in the guanidine hydrochloride solution this approach is shown to be sensitive enough to detect and quantitate ligand induced changes in the strength of subunit interactions.

Supported by NSF Grant MCB 0448905 to Ellis Bell
Controlling a Chemical Reaction with an Aptamer
Authors: Wang Tianjiao , Nilsen‐Hamilton Marit
Published: June 2021

Like enzymes, aptamers provide structurally‐defined binding pockets to hold their target molecules tightly. Unlike ribozymes, aptamers are not catalytic but could be applied to slowing down the rates of reactions that occur spontaneously in an aqueous environment. Examples of such chemical reactions might be the many oxidative reactions that are involved in inflammation, aging and cancer. To suppress such reactions, an aptamer needs to prevent access to or reactivity of the target molecule with very small oxidative species. We demonstrate that a model reaction, decolorization of malachite green (MG) by hydroxyl ions, can be regulated by the interaction of MG with the MG aptamer (MGA). That the effect of the MGA on the rate of MG decolorization is due to the structure of the MGA binding pocket is demonstrated by reversal of the effect by an antisense oligonucleotide to MGA. UV spectroscopy and stop‐flow analysis were used to study the kinetics of MGA suppression of MG decolorization of the MG‐MGA interaction. The 20 min half life of MG decolorization was reduced to almost infinite with a 3‐fold excess of MGA. The half‐life of the MG‐MGA interaction was 1.7 sec. The results show that an aptamer can be used to suppress a spontaneous chemical reaction and that this suppression can be readily controlled by an antisense oligonucleotide. Funded by the DOE under contract W‐7405‐Eng‐82 with the Ames National Laboratories.
The impact of dairy and sweetened beverage consumption on diet quality, nutrient intake, and weight of a multi‐ethnic population of Head Start mothers
Authors: O'Neil Carol E. , Nicklas Theresa A. , Liu Yan , Franklin Frank A.
Published: June 2021

To assess the impact of milk and sweetened beverage (SwB) intake on diet and weight in Head Start mothers, three 24‐hour dietary recalls were collected on 609 Black (43%), Hispanic (33%), or White (24%) women in AL and TX. Women were divided into four beverage consumption groups: low milk/high SwB, high milk/low SwB, low milk/low SwB, & high milk/high SwB. Diet quality was assessed using the mean adequacy ratio (MAR). Analysis of variance was used to detect differences in continuous variables; X² was used for categorical outcomes. Covariates for general linear models were age, ethnicity, body mass index (BMI), and energy intake. MAR was higher in high milk/low SwB (71.9) than low milk/high SwB (58.7). High milk/low SwB had highest the intakes of vitamins A, D, and B6; riboflavin, thiamin, folate, phosphorus, calcium, iron, and potassium. Protein intake was higher in both low SwB groups than in the low milk/high SwB group. Total fat intake was highest in the low milk/low SwB, and lowest in high milk/high SwB. Intake of total and added sugars was higher in both high SwB groups when compared with the two low SwB groups. There was no difference in BMI across the four consumption groups. Overall, milk consumption was low. The high milk/low SwB group had better nutrient intakes, including higher consumption of calcium, potassium, and vitamin A—shortfall nutrients in adults. This work was supported by the USDA and Dairy Management, Inc.
Molecular and cellular effects of α‐, and γ‐tocopheryl phosphate on THP‐1 monocytes
Authors: Zingg Jean‐Marc , Negis Yesim , Gianello Robert , Libinaki Roksan , Ogru Esra , Meydani Mohsen , Azzi Angelo
Published: June 2021

The vitamin E derivative, alpha‐tocopheryl phosphate (αTP), can be isolated from food and animal tissues in amounts of nmol/g of extracted material. Whereas the phosphate ester increases the chemical stability of alpha‐tocopherol (αT), it suppresses its function as a direct free radical scavenger. Nevertheless, in cells TP influences signal transduction and gene expression, possibly by interacting with specific enzyme(s) and/or receptor(s). Small amounts of αTP formed from αT in cultured cells further suggest a physiological role for this compound. We have now compared the inhibitory effects of αTP (acid and sodium salt) and γ‐tocopheryl phosphate (γTP) (acid and sodium salt) on the proliferation of THP‐1 monocytic leukemia cells. αT does not affect the cell proliferation rate in these cells, whereas αTP and more potently γTP inhibit THP‐1 cell proliferation. We found that one or more cell anion transporters are modulating the response of THP‐1 cells to the treatment with αTP and γTP, as determined by using specific transport inhibitors, such as glybenclamide/glyburide. In addition, the effects of these vitamin E derivatives on gene expression are assessed, in particular on the inhibition of CD36 scavenger receptor gene expression. (This study is supported by a Phosphagenics Ltd. fellowship to JMZ, and USDA Contract #58‐1950‐7‐707).
α‐Tocopherol counteracts ritonavir‐induced inflammatory cytokines secretion in THP‐1/macrophage cells
Authors: Guo Weimin , Meydani Mohsen , Azzi Angelo
Published: June 2021

Treatment with protease inhibitor (PI) drugs of HIV‐infected individuals has significantly increased their life span. However, one of the side effects of PI drugs is the development of premature atherosclerosis, whose molecular pathogenesis remains unclear. Earlier, we have reported that α‐tocopherol (α‐T) normalizes CD36 overexpression and reduces oxLDL uptake in THP‐1 cells. Since inflammation is a major player in the pathogenesis of atherosclerosis, we hypothesize that PI drugs, such as ritonavir, increase pro‐inflammatory cytokines synthesis and that α‐T supplementation counteracts this effect by suppressing pro‐inflammatory cytokines synthesis. Here, we report that after differentiating THP‐1 cells to macrophages, ritonavir treatment (10μg/mL) significantly increased expression of pro‐inflammatory cytokines, IL‐6, MCP‐1 and IL‐8, both at mRNA and protein levels. This effect of ritonavir was significantly suppressed by treatment of THP‐1/macrophages with 50μM α‐T. Our data suggest that supplemental α‐T may suppress ritonavir‐induced pro‐inflammatory cytokines in HIV‐infected individuals. (Supported by USDA agreement #58‐1950‐7‐707)
α2A Adrenergic Receptors Modulate CA3‐CA3 Pyramidal Cell Communication in the Rat Hippocampus
Authors: Jurgens Chris WD , Doze Van A
Published: June 2021

The adrenergic system has potent antiepileptic properties and also enhances certain memory processes. The pharmacology and mechanisms underlying these remain unclear. We have identified an α2A adrenergic receptor (α2AAR) which robustly inhibits the initiation of epileptiform activity in the CA3 region of the rat hippocampus, a structure critical for learning and memory and also a common focus for temporal lobe seizures. Using whole‐cell recordings, we examined excitatory post‐synaptic currents evoked from the major CA3 synaptic inputs (mossy fiber and CA3 recurrent), as well as the CA3 inputs onto CA1 pyramidal neurons to further localize this adrenergic effect. Electrophysiological evidence suggests that α2A‐AR activation preferentially inhibits the recurrent hippocampal CA3 glutamatergic synapses compared to the excitatory drive to and from the CA3 cells. Further electrophysiological evidence suggests that this occurs though a presynaptic inhibition of glutamate release. Since overexcitation of the recurrent CA3 synapses is thought to occur in temporal lobe seizures, selective inhibition of presynaptic hippocampal excitatory communication may explain how the adrenergic system may be antiepileptic while at the same time not affect other cognitive functions such as learning or memory. This study was supported in part by the Epilepsy Foundation (CWDJ), North Dakota EPSCoR through NSF grant EPS‐0447679 (VAD), NSF CAREER award 0347259 (VAD), and NIH grant 5P20RR017699 from the COBRE program (VAD).
Inhibition of heat shock protein 90 (Hsp90) and degradation of client proteins associated with oncogenesis using analogs of Novobiocin
Authors: Higgins Michelle , Vielhauer George , Weir Scott , Holzbeierlein Jeff , Blagg Brian
Published: June 2021

Prostate cancer remains a lethal disease with over 30,000 men dying per year. Thus, there is a critical need to develop novel therapies that translate to improved clinical outcomes. The objective of these studies is to design C‐terminal inhibitors of the molecular chaperone hsp90 resulting in client protein degradation and inhibition of multiple signaling pathways causing cancer cell death of in vitro and in vivo. C‐terminal inhibitors of hsp90 were tested in a dose response manner using the androgen independent PC‐3 prostate cancer cell line by Western blot analysis and examined degradation of client proteins (AKT, HER2, and BCL‐2). Additionally, one inhibitor, KU32, was used in an in vivo orthotopic murine tumor model and examined for reduction of prostate serum antigen (PSA) and degradation of client protein in treated and untreated animals. Prostate cancer cells treated with these inhibitors demonstrated the hallmarks of hsp90 inhibition, specifically, the increased expression of hsp90 and decreased expression of client proteins in a dose dependent fashion. The in vivo studies are currently ongoing. These data suggest that C‐terminal inhibitors may debilitate cancer cell function and slow tumor cell proliferation.

These studies were supported by funding from the National Cancer Institute (5 U01 CA120458‐02) and the Department of Defense (W81XWH‐05‐1‐0594).
Serotonin mediated Ca2+ events are reduced in pulmonary arterial myocytes of chronic hypoxic fetal sheep
Authors: Osman Noah J , Goyal Ravi , Pearce William J , Longo Lawrence D , Wilson Sean M
Published: June 2021

Serotonin (5HT) is important to pulmonary arterial (PA) reactivity and alterations in 5HT signaling are implicated in pulmonary hypertension in newborns as well as adults. Several reports also provide evidence for alterations in 5HT mediated contractility with maturation and in response to chronic hypoxia (CH). Further to this 5HT2A receptor activation and Ca2+ signaling is important to the contractility responses induced by 5HT, and we have evidence that Ca2+‐dependent contractility is reduced with CH in PA from sheep fetus. To delineate the cellular mechanisms associated with the loss in Ca2+‐dependent contractility we tested the hypothesis that 5HT generated Ca2+‐signaling is reduced with CH in fetal sheep. This was examined using in‐situ confocal imaging of Flou‐4 in PA isolated from CH fetus and adult. PA myocytes of CH fetal and adult had spontaneous cytosolic Ca2+ elevations and 10 μM 5HT induced cytosolic Ca2+ elevations in myocytes of fetal and adult sheep. The frequency of 5‐HT‐mediated Ca2+ oscillations in responsive cells was 0.01 ± 0.002 Hz in fetus and 0.021 ± 0.002 Hz in adult while the density of cells exhibiting Ca2+ elevations was 0.003 ± 0.001 cells/mm2/sec in fetus and 10‐fold greater in adult, being 0.03 ± 0.003 cells/mm2/sec. The reduced 5HT mediated Ca2+ signaling in fetal cells may underlie the loss of Ca2+‐dependent contractility previously observed. (Support from NSF, NIH and UM)
Acetylcholine receptor‐mediated contractility of ovine pulmonary arteries: Changes with maturation and chronic hypoxia
Authors: Nguyen Dan , Goyal Ravi , Derrick Allison , Loftin Matthew , Osman Noah J , Pearce William J , Longo Lawrence D , Wilson Sean M
Published: June 2021

Muscarinic acetylcholine (ACh) receptor (mAChR) activation relaxes arteries through endothelium‐dependent NO signaling pathways and endothelium removal from most systemic arteries alleviates ACh mediated relaxation. However, Ach induces “paradoxical” contractility in endothelium disrupted pulmonary arteries (PA), through mAChR activation in multiple species including human. Chronic hypoxia (CH) disrupts vascular endothelium, producing loss of endothelium‐dependent PA relaxation. Given the intimacy between endothelial and myocyte function the hypothesis that CH reduces ACh‐dependent PA contractility was tested by performing wire‐myography of endothelium‐denuded PA rings from normoxic fetal and adult animals. The data show that Ach (100 μM) contracted arteries pre‐contracted with 125 mM KCl and this contractility was reduced by CH in adult PA and ablated in fetus. In CH adult, the mAChR agonist carbachol (CCh, 10 μM) caused contraction while 1 μM atropine (mAChR antagonist) blocked Ach and CCh contractility. Consistent with mAChR activation, In situ confocal microscopy approaches show Ach caused cytosolic Ca2+ increases in PA myocytes of CH adult. In conclusion, this study provides evidence for CH induced loss of mAChR PA contractility, a process that likely helps match ventilation to perfusion, especially as ACh causes bronchoconstriction. (Support from NIH, NSF and UM)
Single fiber muscle contractile properties in mobility‐limited older adults
Authors: Fielding Roger A. , Krivickas Lisa S. , Gavin Brian P. , Reid Kieran F. , Carabello Robert J. , Phillips Edward M. , Frontera Walter R.
Published: June 2021

Mobility limitations in older adults are associated with reductions in whole muscle size, strength and power. We examined whether alterations in isolated single muscle fiber contractile properties contribute to the observed reductions in whole muscle function in older adults. Percutaneous needle biopsies of the vastus lateralis muscle were obtained from 20 middle aged healthy (MH, 40‐55y, 9 male), 19 older healthy (OH, 70‐85y, 15 male), and 13 older mobility‐limited (OML, 70‐85y, 7 male, SPPB ≤9) adults. Contractile properties of permeabilized single muscle fibers were evaluated. Data from isolated type I fibers revealed no significant differences in force or power generating properties from muscle obtained from MH, OH, or OML.

These data suggest that differences in type I muscle fiber contractile properties cannot explain the deficits in force and power generating capacity observed in mobility‐limited older adults.

Supported by USDA 58‐1950‐7‐707 and NIA RO1‐AG‐18844
Functional topography of the dorsomedial hypothalamus
Authors: McAllen Robin Michael , Tanaka Mutsumi
Published: June 2021

The dorsomedial hypothalamus (DMH) has been proposed to mediate, perhaps integrate, autonomic components of both the defense reaction to acute stress and thermoregulatory responses to cold. We made a finer‐grained functional map of the DMH than achieved hitherto, to see whether ‘cold’ and ‘defense’ patterns might be generated by distinct DMH cell groups. We simultaneously recorded phrenic nerve activity and single cutaneous vasoconstrictor (CVC) fibers supplying plantar skin in 14 vagotomized, ventilated, urethane‐anesthetized rats (1–1.4 g/kg, i.v.). To characterize the response to ambient cooling, we cooled the trunk skin by a water jacket: this reversibly activated plantar CVC fibers and depressed phrenic burst rate. D,L‐homocysteic acid (DLH; 15 nl, 50 mM) microinjected into 76 DMH sites to activate small groups of cell bodies never reduced phrenic activity. Increased phrenic burst rate was evoked most strongly from caudal regions, plantar CVC activity from rostral regions: the ratio of the two responses varied systematically with location. In paired comparisons they were uncorrelated. Partly overlapping but distinct contour maps of response magnitude could be plotted over the DMH region for phrenic rate, CVC fibers and heart rate. These findings indicate that DMH neurons show functional topography but limited capacity to integrate autonomic/respiratory motor patterns. Supported by ARC.
Erythropoietin (EPO) and Soluble Traansferrin Receptor (sTfR) Responses at 4300 m Before and After Normobaric Intermittent Hypoxic Exposure
Authors: Staab Janet E. , Muza S. R. , Beidleman B. A. , Fulco C. S. , Staab J. S. , Jones J. E. , Reese M. L. , Cymerman A.
Published: June 2021

During continuous altitude exposure EPO initially rises then falls while sTfR rises progressively. The purpose of this study was to determine if one week of daily 3‐h normobaric intermittent hypoxic exposures (IHE) (2h PIO2=90mmHg rest, 1 h PIO2=110 mmHg exercise) would elicit the same EPO and sTfR responses observed during continuous altitude exposure. Fifteen sea‐level (SL) resident men (mean±SD;21±6 yr) were exposed to 4300 m in a hypobaric chamber (preIHE) followed by ~12 days at SL. Then, for 6–7 d, nine received IHE treatment (T‐IHE) and six received a sham IHE treatment (S‐IHE) followed by 2.5 d of no IHE before the 2nd 4300 m exposure (postIHE). Resting SaO2 was measured during IHE by pulse oximetry. Serum EPO and sTfR (ELISA) were measured at SL and 24 h into each 4300 m exposure. Over ~7 d of IHE, SaO2 during T‐IHE increased (p<0.01) from 75±4% to 81±5% while SaO2 during S‐IHE remained at 98±1%. EPO (mIU/ml) increased (p<0.01) similarly for T‐IHE and S‐IHE from SL (8±1 vs. 6±1) to 4300 m during both PreIHE (143±38 vs. 146±46) and PostIHE (132±33 vs. 120±41). There was no change in EPO from PreIHE to PostIHE. sTfR (nmol/L) did not change for T‐IHE or S‐IHE from SL (17±1 vs. 18±1) to PreIHE (18±1 vs. 20±1) or PostIHE (18±1 vs. 18±1). Thus, 6–7 d of IHE did not elicit similar responses in EPO and sTfr as observed during continuous altitude exposure. Funding provided by USAMRMC. Authors’ views; not official U.S. Army or DoD policy.
Oxygen Dependency of Hydrogen Sulfide‐mediated Vasoconstriction in Cyclostome Aortas
Authors: Olson Kenneth R. , Forgan Leonard G. , Dombkowski Ryan A. , Forster Malcolm E.
Published: June 2021

Hydrogen sulfide (H2S) is a vasoregulatory molecule and recent evidence suggests it may be an O2 sensor for hypoxic vasoconstriction (HVC). Here we examined H2S ‐ O2 interactions in hagfish vessels. In myographic studies, H2S (Na2S) dose‐dependently constricted dorsal aortas (DA) and efferent branchial arteries (EBA) but did affect ventral aorta or afferent branchial arteries; hypoxia produces similar responses. Hypoxia enhanced DA sensitivity to H2S. HVC in DA was augmented by the H2S precursor cysteine and inhibited by aminooxy acetate (AOA), an inhibitor of the H2S‐synthesizing enzyme, CBS. HVC was unaffected by propargyl glycine, the CSE inhibitor. DA O2 consumption (MO2) was constant between PO2 15–115 mmHg, but decreased when PO2 <15 mmHg and increased after PO2 exceeded 115 mmHg. 10 uM H2S increased MO2, whereas MO2 fell when [H2S] >100 uM. Consistent with the effects on HVC, cysteine increased and AOA decreased MO2. These results show that H2S is a monophasic vasoconstrictor of specific cyclostome vessels and because hagfish lack vascular NO, and vascular sensitivity was enhanced at low PO2, it is unlikely that H2S contractions are mediated by either H2S‐NO interaction or an oxidation product of H2S. They also support the hypothesis that H2S metabolism is an O2 sensor in vertebrate vascular smooth muscle. Support: NSF IBN 0235223, IOS 0641436 and Erskin Fellowship (KRO)
Catecholamine (CA) content and release from adrenal chromaffin cells is differentially influenced by fatty acids.
Authors: Ribeiro Laura Teixeira , Martel Fátima , Azevedo Isabel
Published: June 2021

This study aimed to investigate the effects of several fatty acids on CA handling by bovine chromaffin cells. Cells were incubated for 24 h with ethanol (control), butyric acid (BA; 100 μM‐1 mM), capric acid (CAP; 50–100 μM), palmitic acid (PAL; 50–100 μM) or arachidonic acid (AA; 50–100 μM). Afterwards, for studies on CArelease, cells were preincubated for 10 min, and then for 15 min with or without KCl. Under control conditions, the average cellular content was found to be 413 ± 19 and 202 ± 10 nmoL/mg protein for adrenaline (AD) and noradrenaline (NA), respectively. Basal release (expressed as % of the total amine content), during 10 min of preincubation, was 8 ± 0.4 and 9 ± 0.5 for AD and NA, respectively. With the exception of BA, all the fatty acids induced a significant reduction of both AD and NA cellular content, as well as of total CA production. This significantly lower CA cellular content observed after 24 h exposure of cells to AA or CAP was accompanied by a tendency to a higher CA basal release and a lower CA release induced by KCl. In conclusion, fatty acids differentially influence CA handling by adrenal chromaffin cells. Altogether the data are compatible with a reduction of the vesicular CA transporter function due to the long‐term exposure to some of the fatty acids. Supported by FCT (SFRH/BPD/28597).
Does L‐leucine use a dipeptide transport protein for absorption by crustacean intestine?
Authors: Mullins Anna L. , Ahearn Gregory A.
Published: June 2021

Previous work with mammals and crustaceans has shown that L‐histidine forms a bis‐complex with zinc ([His]‐Zn‐[His]) in solution. A PEPT‐1‐like dipeptide carrier was suggested as a transport mechanism by which the bis‐complex would pass across the intestinal luminal membrane of the lobster, Homarus americanus. In addition, this early work showed that L‐leucine inhibited transport of the histidine bis‐complex. In the present study, lobster intestines were isolated and mounted in a perfusion chamber in order to determine how 3H‐L‐leucine is transported across lobster intestine in the presence of zinc and to deduce whether 3H‐L‐leucine and L‐histidine are using the same transport system. It was found that 3H‐L‐leucine transport was a hyperbolic function of luminal zinc concentration. In addition, transmural M to S (mucosa to serosa) 3H‐L‐leucine transport in physiological saline with 25 μM Zn was stimulated by decreasing the pH from 7.1 to 6.0. Transmural M to S 3H‐L‐leucine fluxes (0.5 to 10 mM) in physiological saline at pH 5.5 and 25 uM Zn displayed sigmoidicity (Hill Coefficient ≈ 2.0). If leucine was transported by a PEPT‐1‐like transporter, the amino acid may form a bis‐complex with zinc in solution (two amino acids coupled with a zinc ion) and its transport would be sigmoidal, displaying a Hill coefficient of 2. Finally, it was found that luminal zinc increased the net M to S flux of 3H‐L‐leucine across the intestine. These experiments support the hypothesis that L‐leucine forms bis‐complexes with zinc ([Leu]‐Zn‐[Leu]) and uses a PEPT‐1‐like protein system for transport across lobster intestine. Supported by NSF grant IBN04‐21986.
Diurnal pattern in endogenous insulin secretion persists in severely injured patients
Authors: Pidcoke Heather F , Wolf Steven E , Jones John A , Holcomb John B , Wade Charles E
Published: June 2021

Intensive insulin therapy decreases mortality due to critical illness but glucose and insulin variability pose a challenge to tight glucose control. We previously demonstrated that circadian rhythms in glucose and exogenous insulin requirement are preserved, and hypothesized that similar patterns in endogenous insulin secretion are also present.

Patients in burn and surgical ICUs at a single center receiving intensive insulin therapy were admitted to the study. Blood samples were collected at 4 am and 5 pm. An enzyme‐linked immunosorbent assay (ELISA) for C‐peptide quantified endogenous insulin. Data were analyzed with paired Student's t‐test.

C‐peptide levels from fourteen patients admitted to the study were averaged (mean #values/subject was 30±31, range: 6–125). Morning C‐peptide levels (5.1 ng/mL ± 3.4 ng/mL, range: 1.6–11.3 ng/mL) were lower compared to evening levels (6.5 ng/mL ± 4.2 ng/mL, range: 1.7–15.9 ng/mL, p=0.016).

Endogenous insulin secretion demonstrates diurnal variation in critically injured patients, with higher evening levels relative to morning values. This study contributes further evidence that the circadian rhythms in hormones of glycemic regulation are preserved after severe trauma. Performance of intensive insulin protocols may improve if glucose targets are adjusted to reflect 24 hour periodicity. Grant support from the NIH, JDRF, and MRMC.
Immunohistochemical Demonstration of Avian Multi‐drug Resistance Protein 4 (Mrp4) in Renal Proximal Tubule Epithelium
Authors: Coleman Amy Michele , Renfro J. Larry
Published: June 2021

Birds are uricotelic and most of the urate is excreted by active tubular secretion into the urine. The mechanism of urate transport across the apical membrane of the intact proximal tubule epithelium during secretion is uncertain. Our previous studies of urate transport have implicated Mrp4 as a dominant pathway for urate secretion across the apical membrane. Short hairpin RNA interference (shRNAi) effectively knocked down expression of Mrp4 mRNA in chick renal proximal tubule epithelial cell primary cultures (PTCs) and reduced transepithelial urate secretion approximately 80%. Here, we report the production of a rabbit custom polyclonal antibody designed against chicken Mrp4 (Genscript). Immunoblots of isolated brush border membrane samples from chicken kidney and allyl alcohol‐stimulated mouse liver samples confirmed the labeling of Mrp4 by the antibody (~ 150 kDa). Antigenicity was not detected in isolated basolateral membrane vesicles of chick kidney. Fluorescence immunohistochemistry and confocal microscopy of intact renal proximal tubules and PTCs showed localization of Mrp4 to the apical membrane of the cells and confirmed the lack of Mrp4 in the basolateral membranes. In PTCs where shRNAi was introduced to knockdown expression of Mrp4 mRNA, immunohistochemical fluorescence microscopy confirmed a substantial reduction of Mrp4 protein in the apical membrane microvilli at seven days post‐transfection. Supported by NSF.
Dual lumen catheter for heparin‐free hemorrhage in rats: design, construction, and use
Authors: Atkins James , Lee Chae‐Hyuk , Barrows Elizabeth , Quinn Peter , Gonzales Raoul , Oliver James
Published: June 2021

Intermittent blood withdrawal is required for most studies of hemorrhagic shock; however, these experiments are often complicated by clot formation within the withdrawal catheter. In order to avoid the systemic administration of heparin and we sought to construct a dual lumen catheter that would allow for regional anticoagulation within the withdrawal catheter. The free end of the outer catheter was connected to a computer controlled withdrawal pump while the inner catheter was connected to a similarly controlled infusion pump. Whenever blood was withdrawn through the outer catheter, citrate was infused into the inner catheter at fixed volume ratio of 1:9 infusion:withdrawal. In rodent hemorrhage experiments, the dual lumen catheter was able to successfully bleed the animal to hemodynamic decompensation (avg. volume = 9.5 ± 2.9 ml) without clotting. The ionized calcium of the rats did not change beyond that which would be normally expected from severe hemorrhage. (1.21 ±0.07 mM control vs 1.08 ± 0.18 after hemorrhage). Prothrombin times were not altered during hemorrhage. We conclude that a dual‐lumen catheter using regional citrate anticoagulation can be used as a means of achieving heparin‐free hemorrhage of rodents. The design of the catheter prevents systemic effects of the citrate on the animal and could potentially be used with other anticoagulants. Funded by US Army Medical Research and Materiel Command.
Rhythmic contractions in the portal “corkscrew” vein of a colubrid snake
Authors: Conklin Daniel J. , Bishop B. , Hargens A. , Olson K. , Lillywhite H.
Published: June 2021

Terrestrial, and especially scansorial, species of snakes have portal veins with a corkscrew morphology immediately posteriorad of the liver. A corkscrew vein can act as a functional valve that impedes gravitational shifts of blood volume. In the corkscrew portal vein of a colubrid snake, spontaneous low frequency and low amplitude contractile rhythms were observed in 11 % of all vein rings. Catecholamines stimulated dose‐dependent cyclical contractions in 89 % of portal vein rings, but not in abdominal veins. Increased contractile amplitude and cycle frequency were stimulated by catecholamines, angiotensin II, and stretch. Near maximal agonist‐induced tension eliminated cyclical contractions, which resumed after removing agonists or adding acetylcholine, but were unaffected by adrenergic receptor antagonists. Treatment with ouabain, verapamil, or indomethacin but not tetrodotoxin rapidly inhibited cyclical contractions, indicating dependence on Na+‐K+ ATPase, extracellular Ca2+, and prostanoids. Histologically, portal vein rings had two smooth muscle layers (luminal, circular; adventitial, longitudinal) sandwiched on a collagen layer. These data suggest that portal vein rhythmic contractility and corkscrew morphology act in concert to improve venous return and to mitigate blood pooling in snakes. Supported by NSF IBN 91‐05247 (KRO), NASA 199‐14‐12‐04 (ARH), and NRC (SRA to HBL).
Removal of cardiac shunt impairs growth and causes ventricular hypertrophy in American alligator
Authors: Eme John , Gwalthney June , Owerkowicz Tomasz , Blank Jason M. , Hicks James W.
Published: June 2021

Reptilian cardiac shunting is hypothesized to confer a fitness advantage via physiological functions including inducing hypometabolism, aiding recovery from metabolic acidosis and facilitating digestion. Elimination of cardiac shunt has not previously been shown to reduce fitness. Crocodilians possess a unique reptilian cardiac anatomy (fully‐divided ventricle) that allows surgical elimination of right‐to‐left cardiac shunt. We altered the outflow tract of hatchling alligators by occluding the left aorta proximal and distal to the foramen of Panizza. Surgical and sham groups (n=36 each) were subsequently split into three exercise regimes: run, swim and sedentary (n=24 each). Run and swim groups were exercised to exhaustion using a treadmill or swim flume every other day for 15 months, and growth was monitored. Elimination of cardiac shunt led to significantly lower mean body mass of surgical alligators (~10%), but resulted in a significantly larger ventricular mass (~65%). Maximum aerobic capacity of alligators was increased (~15%) by exercise training (both running and swimming), but not affected by shunt elimination. We suggest cardiac shunting has adaptive significance to crocodilians by allowing faster growth and reducing cardiac work. Funded by NSF IOB 04445680 to JWH.
Does aerobic dive capacity constrain winter diving behavior in crabeater seals?
Authors: Burns Jennifer M , Costa Daniel P
Published: June 2021

During austral summers, crabeater seals (Lobodon carcinophagus) preferentially target the large zooplankton and krill that are abundant in surface waters. However, during winter months, seals forage deeper and closer to the seafloor, where their preferred prey aggregate. As a result, winter foraging is associated with a much greater proportion of dives longer than the seals’ calculated aerobic dive limit (cADL, 6.1 ‐ 11.8 min). To determine if aerobic capacity constrains winter foraging activities, we examined the relationship between the cADL and dive depth, bottom time, and bathymetric depth in 34 crabeater seals ranging in mass from 118 – 365 kg. While fewer than 10% of dives shallower than 150 m were anaerobic, this percentage exceeded 30% for dives deeper than 250 m. These deeper dives were most common in late winter and during the day. Interestingly, larger animals made more anaerobic dives, perhaps because they dove closer to the seafloor than smaller animals. In combination, our findings suggest that body size and physiological capacity influence crabeater seal foraging strategies, particularly during winter. These findings have implications for crabeater seal foraging success, particularly if warming along the Antarctic Peninsula causes changes in the abundance and depth distribution of preferred prey items. This research was funded by NSF OPP # 0003956 & 0006429 to JMB and DPC.
Moderate hypoxia (10 kPa) extends lifespan with adult but not juvenile exposure in the fruitfly, Drosophila melanogaster
Authors: Harrison Jon Fewell , Rascon Brenda
Published: June 2021

Oxygen provides the substrate for most ATP production but also serves as the source of reactive oxygen species that promote aging. Multiple invertebrate studies have shown that pure oxygen atmospheres strongly reduce life span, but it is unknown whether the relationship between oxygen level and life span is linear across varying atmospheric pO2 levels. In this study, we reared Drosophila melanogaster at five adult oxygen levels (2, 10, 21, 40 or 100 pO2) in order to investigate the relationship between atmospheric oxygen and life span. We show that that the response of fruit fly life span to ambient oxygen is parabolic, with, reduced longevity at higher and lower oxygen levels; the optimal pO2 for longevity is moderately hypoxic (10 kPa). We also reared D. melanogaster at three juvenile pO2 levels, to test whether juvenile exposure to hypoxia or hyperoxia leads to compensatory responses that enhance the ability of flies to cope with high or low oxygen as adults. In general, there was little evidence for beneficial plasticity, and most of the data suggested that exposure to hypoxia or hyperoxia as juveniles lead to damage that exacerbated effects of non‐optimal oxygen levels in adults. Supported by NSF IOB 0419704 to JFH and NSF HRD‐0114712 to WAESO at ASU.