bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Role of Hyperpolarization‐Activated Cyclic Nucleotide‐Gated Channels in Ileus Development
Authors: Gergely Bence , Sipos Ádám , Docsa Tibor , Uray Karen 
Published: April 2020

IntroductionIleus, a decrease or cessation of intestinal contractile activity, affects millions of trauma, post‐operative, and critically ill patients, resulting in longer hospital and ICU stays and increased patient complications. Mechanosensitive ion channels play a role in both the physiological and pathological responses to mechanical stimuli. We found that hyperpolarization‐activated cyclic nucleotide‐gated channel 2 (HCN2) was down‐regulated in edematous intestinal smooth muscle layers. However, the effects of altered HCN channel activity on intestinal contractile activity are largely unknown.ObjectiveThe purpose of the study was to investigate the effects of HCN inhibition on intestinal contractile activity. We hypothesize that inhibition of HCN will decrease intestinal contractile activity. Furthermore, we hypothesize that HCN activity is inhibited by mechanical stretch and/or inflammatory mediator changes that occur during the development of ileus.MethodsThe effects of HCN inhibitors on intestinal contractile activity were investigated ex vivo in an organ bath system. The effects of mechanical stretch, as would occur during the development of ileus, were subsequently examined. The effects of CXCL1, an inflammatory mediator shown to increase in both animal models of ileus and in trauma patients with ileus, were also examined.ResultsHCN inhibition with ZD7288 suppressed agonist‐induced intestinal contractile activity in a dose‐dependent, tetrodotoxin‐resistant manner. Inhibition with an alternative HCN inhibitor, Zatebradine, inhibited agonist‐induced contractile activity in a similar manner. HCN inhibition suppressed calcium sensitivity in the small intestinal. Interestingly, HCN inhibition did not inhibit spontaneous contractile activity. Subjecting intestinal tissue to increased stretch or pretreatment with CXCL1 reduced the effects of ZD7288 on intestinal contractile activity, suggesting that both mechanical stretch and inflammation reduce HCN activity. Preliminary data show that increased intestinal wall stretch decreased HCN protein levels.ConclusionsThe effects of HCN inhibition are tetrodotoxin‐resistant; thus, the effects of HCN inhibition are mediated in the smooth muscle, not the enteric nervous system. Both mechanical stretch and inflammation, which occur during the development of ileus, can inhibit HCN activity. Taken together, these data show that HCN channels are important in agonist‐induced intestinal contractile activity. HCN channels are a novel potential target in the treatment of ileus in post‐operative or critically ill patients.Support or Funding InformationHungarian National Research, Development, and Innovation Office (NKFI 120669) and Institutional Developments for Enhancing Intelligent Specialization Grant (EFOP‐3.6.2‐16‐2017‐0006)
Hypergravity Reduces Astrocyte Migration by Altering Cytoskeletal Dynamics
Authors: Liemersdorf Christian , Lichterfeld Yannick , Kalinski Laura , Frett Timo , Jordan Jens , Hemmersbach Ruth 
Published: April 2020

Glial scar formation through astrocytes impairs neural regeneration following spinal cord injury, head trauma, or stroke. Astrocyte migration towards the lesion and induction of a reactive astroglial phenotype require dynamic cytoskeletal protein rearrangements. In this glial cell model, hypergravity was used to alter cytoskeletal dynamics, i.e. stabilizing microtubules while destabilizing actin filaments. We hypothesize that increased gravitational (mechanical) loading by means of centrifugation (hypergravity) modulates in vitro astrocyte function in a way that could reduce their potential for scar formation.We exposed primary murine cortical astrocytes to 2g using two types of custom‐designed hypergravity platforms at DLR (Cologne, Germany) and assessed a variety of parameters important for glial scarring in vitro. The platforms unlike commercial laboratory centrifuges model physiological hypergravity and allow for cell cultivation and live‐cell imaging. Primary astrocytes were isolated from wildtype (C57BL/6J) as well as transgenic LifeAct‐GFP mice and subjected to increased gravitational load. We measured cell proliferation and survival, after 7 days of exposure to 2g, as well as spreading and migration rate online for 24h. We visualized morphological features, cytoskeletal actin filament dynamics, reactivity markers and investigated expression levels of focal adhesion‐related proteins.The exposure to 2g hypergravity induced a decrease in cell spreading (20%) coincidental with an inhibited migratory behavior (40%) and altered cytoskeletal dynamics. Astrocytic proliferation and survival were not affected. The expression of the focal adhesion marker vinculin was increased by 70–80%.We conclude that hypergravity attenuates astrocyte spreading and migration. These parameters are crucial for glial scar formation, while basic cellular processes, such as proliferation and apoptosis were unchanged. The response appears to be mediated through altered cytoskeletal dynamics and may provide targets for therapies promoting neuronal regeneration.
Unraveling Intron Retention: Differential Expression Pattern in Brain and Kidney Cells After Hypoxia or Ischemia
Authors: Northrup Victoria , Perez Lester J. , Edgett Brittany A. , Simpson Jeremy A. , Brunt Keith R. 
Published: April 2020

IntroductionThe inclusion of introns in the mature mRNA (intron retention), has been recently uncovered as a mechanism for the regulation of genetic expression in vertebrates. Intron retention is implicated in tissue specific protein diversity, splicing regulation and control of gene expression. We have recently identified intron retention as a mechanism involved in the regulation of erythropoietin (EPO). EPO is the key regulator of erythropoiesis and is transcriptionally regulated by hypoxia inducible factor 2 (HIF2). The ability to easily detect intron retention could add to our ability to better understand genetic regulation of our genomes in stress conditions, such as ischemic diseases (ex/ myocardial infarction (MI) or stroke).ObjectiveTo evaluate the role of intron retention mechanism in EPO expression level to gain a better understanding of its regulation in response to hypoxia and ischemia, conditions which result in EPO expression.MethodsHTB16 (neuroblastoma), CHP212 (glioblastoma), hCMEC/D3 (blood brain barrier) and HEK293 (kidney) were placed in hypoxic (1% oxygen), ischemic (1% oxygen, no serum or glucose) or normoxic conditions (20% oxygen) for 24 hours prior to RNA extraction. Quantitative polymerase chain reaction (qPCR) was performed with two sets of primers for EPO, the first set was targeting intron inclusion with each primer in a separate exon (intron primers) and the second set was targeting a region that spans two exons, which would not amplify if introns were present (non‐introns). Relative quantification for the level of expression was determined by qPCR using β‐actin and YWHAZ as reference genes for normalization purposes. Statistical analysis of transcript expression and the ratio between intron and non‐intron detecting primer sets were determined using analysis of variance (ANOVA).ResultsEPO mRNA level was increased in HTB16, CHP212 and HEK293, but not in hCMEC/D3 cell lines in response to ischemia, but not hypoxia. However, when the ration of intron vs. non‐intron expression level was determined a trend towards more intron retaining transcripts in HTB16, CHP212 and hCMEC/D3 but not in HEK293 was observed, suggesting an induction for the expression of this gene under this ischemic condition.ConclusionsIntron retention mechanism in EPO is taking place in brain‐derived cell lines, but not in kidney‐derived cells. These results indicate a tissue specificity of intron retention mechanism that could regulate differential expression level of EPO under different physiological and stressful conditions. These findings highlight the complexity of EPO regulation. Hence, intron retention must be considered when examining the expression of a gene to better understand expression in ischemic disease models, such as MI or stroke.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada (NSERC)
Effect of Natural Menopause and Hormone Therapy on Markers of Cellular Senescence
Authors: Faubion Laura , White Thomas A. , Peterson Brett J. , Singh Atul K. , Mielke Michelle M. , Miller Virginia M. 
Published: April 2020

BackgroundAccumulation of senescent cells and the release of proteins from those cells contribute to aging‐related disorders. Estrogen may reduce physiological changes associated with aging through inhibition of cell senescence. This study aimed to determine the effects of menopausal hormone treatments on circulating levels of cell senescence markers.MethodsGDF 15, TNF R1, FAS, and MIP1α were measured in serum using bead‐based assays and compared among women who were within 3 years of natural menopause (mean age 52 years) participating in the Kronos Early Estrogen Prevention Study (KEEPS). Women were randomized to either placebo (n=38), oral conjugated equine estrogen (oCEE, n=37), or transdermal 17β‐estradiol (tE2, n=34). Serum levels of the senescent markers for each treatment group were compared to placebo 36 months after randomization using the Wilcoxon rank‐sum test.ResultsSerum levels of GDF 15, TNF R1, and FAS but not MIP1α were lower in both the oCEE and tE2 groups compared to placebo. However, the difference in levels between treatment and placebo were greater for oCEE [−108 pg/mL (p=0.008), −234 pg/mL (p=0.0006), and −1374 pg/mL (p<.0001), respectively] than for tE2 [−76 pg/mL (p=0.072), −105 pg/mL (p=0.076), and −695 pg/mL (p=0.036), respectively].ConclusionsIn recently menopausal women, oCEE and tE2 reduced circulating levels of some markers of cell senescence. Differences in the magnitude of effect may reflect the differences in circulating levels of 17β estradiol, estrone, and sulfonated estrogens between the two active treatments.Support or Funding InformationThe research is supported by the Kronos Early Estrogen Prevention Study, NIA U54‐AG44170, and the Mayo Fnd.
The Influence of Healthy Aging on K+ Channel‐Dependent Microvascular Dilatory Function
Authors: Serviente Corinna , Berry Craig , Kenney W. Larry , Alexander Lacy M. 
Published: April 2020

Aging is associated with reduced microvascular endothelial function, contributing to increased cardiovascular disease risk. Microvascular dysfunction is usually measured by evaluating endothelial nitric oxide‐dependent vasodilation. However, other vascular signaling pathways, including endothelial derived hyperpolarizing factors (EDHFs), acting through K+ channels, are also important for maintenance of vascular health and may be influenced by age and disease. It remains unclear how microvascular EDHFs are affected by healthy aging.PURPOSETo evaluate the microvascular endothelial reliance on K+ channel‐mediated dilation with healthy aging.METHODSHealthy younger (n=6, 27±2 yrs, 23.4±0.5 kg/m2, 40.9±3.9 ml/kg/min) and older (n=9, 64±2 yrs, 23.7±1.1 kg/m2, 37.5±4.0 ml/kg/min) men and women (younger: 3M/3W, older: 3M/6W) underwent graded infusions of the endothelial dependent agonist acetylcholine (ACh, 10−6 to 10−2 M) alone and in combination with the K+ channel blocker tetraethylammonium (TEA, 25 mM) via cutaneous intradermal microdialysis. Additionally, responses to graded infusions (20mM to 100mM) of the K+ channel activator Na2S were evaluated (older, n=10). Red blood cell flux was measured at each site using laser Doppler flowmetry. Cutaneous vascular conductance (CVC: flux/mean arterial pressure) was calculated as a percentage of site specific maximum (43°C + 28mM sodium nitroprusside). Data are presented as mean ± SEM.RESULTSThere was a main effect of age (p<0.001), such that older adults had reduced overall %CVCmax compared to younger adults. There was an interaction between site treatment and age (p<0.001). Within older adults, blood flow was lower in the TEA vs. the ACh site starting at 10−5 M (ACh: 31.8±4.1%CVCmax vs. TEA: 13.2±4.1%CVCmax, p=0.0031), while differences were not observed in younger adults until 10−4 M (ACh: 51.5±5.0%CVCmax vs. TEA: 35.5±5.0%CVCmax, p=0.028). In response to Na2S, there was a main effect of age (p=0.032), with older adults showing overall higher %CVCmax compared to younger adults (older: 56.1±4.4%CVCmax vs. younger 38.8±5.7%CVCmax).CONCLUSIONSThese data suggest that heathy older adults have increased reliance on K+ channel‐dependent endothelial dilatory mechanisms compared to younger adults, suggesting increased reliance on EDHFs with healthy age.Support or Funding InformationNIA T32‐AG049676
Insights into SARM1’s Enzymatic Mechanisms for Executing Pathological Neuronal Death
Authors: Loring Heather , Thompson Paul 
Published: April 2020

The novel NAD+ glycohydrolase, sterile alpha and toll/interleukin receptor (TIR) motif‐containing protein 1 (SARM1), is an active executioner in progressive axonal and neuronal degeneration. This type of degeneration, termed Wallerian degeneration, defines a number of diseases, including neuropathies, traumatic brain injury and neurodegenerative diseases. However, no therapies exist to mitigate these diseases. In fact, prior to the recent discovery of SARM1’s role in triggering Wallerian degeneration, the process was believed to occur passively. Stimulation of SARM1 activates its intrinsic NAD+ hydrolase activity, resulting in NAD+ cleavage and neuronal death. By contrast, knock down or knock out of SARM1 delays degeneration and has shown therapeutic efficacy for various Wallerian‐type diseases, including peripheral neuropathy and traumatic brain injury. NAD+ supplementation alone is also sufficient to delay degeneration, indicating that inhibiting SARM1‐mediated NAD+ hydrolysis would be efficacious. Thus, SARM1’s pivotal role in Wallerian degeneration makes it an attractive therapeutic target that could prevent disease progression. However, the design of therapeutics targeting SARM1 is limited by the dearth of knowledge surrounding its inherent NAD+ hydrolase activity. Illuminating this black box has also been hindered by technical difficulties in obtaining pure active protein. Recently, we were able to purify active bacterially expressed protein, yielding insights into SARM1 morphology and cellular mechanism. Furthermore, we have performed detailed kinetic, catalytic and inhibitory studies, to determine SARM1’s kinetic and catalytic mechanisms and identify potential inhibitors to block SARM1‐mediated cell death. The results of these analyses will be discussed.Support or Funding InformationNIH NINDS F31‐NS‐108610‐01
Sex Differences and Inactivity In Rats Determine the Progression of Increased Sympathetic Activity Over Time.
Authors: Morrison Paul , Tan Can , Draghici Adina , Mueller Patrick 
Published: April 2020

Cardiovascular disease (CVD) is the leading cause of death, with males having a higher risk than females; that is, until menopause, when the risks reverse. Risk factors for CVD include a sedentary lifestyle as well as elevated sympathetic nerve activity (SNA). In women, resting SNA is influenced by the menstrual cycle; however, the interaction between sedentary conditions and the presence or absence of the female reproductive cycle on baseline sympathetic nerve activity is unknown. Therefore, the purpose of this study was to determine the effects of sedentary versus active conditions on resting SNA in male and female rats over time. In particular, we studied rats at four and eight weeks of age (i.e. before and after the onset of the estrous cycle, respectively in female rats). We hypothesized that time‐dependent increases in SNA in sedentary male rats would be offset by four weeks of voluntary exercise (running wheels) and attenuated in sedentary female rats due to the onset of the estrous cycle. We recorded splanchnic SNA from 32 rats under Inactin anesthesia and determined SNA action potential frequency according to Tan et al., (2009). Using a two‐way analysis of variance (ANOVA), with sex and condition as main effects, we found that four week‐old male rats exhibited lower SNA compared to eight week‐old sedentary males (42.0 ±6.3 Hz vs. 62.4 ±5.8 Hz; p=0.015; n=6 each), while exhibiting no significant difference from eight week‐old active males (37.4 ±6.1 Hz; p=0.003; n=7). In contrast, SNA was not significantly different between four week‐old females (54.6 ±4.5 Hz; n=4) and eight week‐old sedentary females (46.0 ±4.3 Hz; p=0.348; n=5), suggesting a lack of inactivity‐related increases in SNA as observed in males. SNA in four week‐old females was also not significantly different than eight week‐old active females (51.0 ±5.8 Hz; p=0.708; n=4). Our results suggest a sedentary lifestyle promotes increases in SNA over time in males but not females, due to physical inactivity in males and a protective effect of ovarian hormones in females. Male sex‐ and inactivity‐dependent increases in SNA likely play an important role in the higher risks of CVD observed in males compared to females.Support or Funding Information(NIH HL096787‐08; AHA25810010)
GPCR Kinase 2 (GRK2) Phosphosphorylation of the Alpha 2A Adrenergic Receptor in Intact Cells
Authors: Sterne-Marr Rachel , Barnett Jessica , Hebert Aaron , Weldin Erica , Farina Zachary , Guirguis Jeska , Varney Matthew 
Published: April 2020

The α2A‐adrenergic receptor (α2A‐AR) is activated by the catecholamine norepinephrine and plays a variety of roles including neuromodulation, vasoconstriction in the cardiovascular system, regulation of insulin secretion and platelet aggregation. Platelet α2A‐AR serves as an excellent substrate for agonist‐dependent phosphorylation by G protein‐coupled receptor (GPCR) kinase 2 (GRK2) in vitro. α2A‐AR is structurally distinct from the well‐characterized β2‐adrenergic receptor (β2AR) in possessing a large (~100 amino acid) third intracellular loop containing four successive serine residues that serve as phospho‐acceptors. When overexpressed with GRK2, the receptor is phosphorylated at all 4 phospho‐sites. Our long‐term goal is to understand how GRK2 interacts with and is activated by GPCRs. We have mapped residues on GRK2 that are required for phosphorylation of β2AR and recruitment to α2A‐AR in intact cells. We wish to assay phosphorylation of α2A‐AR in intact cells and because no commercially available α2A‐AR phosphorylation site antibodies currently exist, we set out to develop such a reagent. An antibody that recognizes α2A‐AR phosphorylation at serines 296–299 was generated and characterized. This antibody detects an epinephrine‐stimulated signal that is increased two‐fold by transfection of GRK2 in COS‐7 cells. We also found that the α2A‐AR was phosphorylated relatively well in the absence of epinephrine and transfected GRK2 suggesting that α2A‐AR is a good substrate for an endogenous COS‐7 cell kinase. Mutagenesis of the receptor has allowed us to measure agonist‐ and GRK‐dependent phosphorylation of α2A‐AR. We are now in a position to characterize GRK2 phosphorylation of this receptor.Support or Funding InformationThis work was funded by the National Science Foundation MCB0744739 and Siena College Center for Undergraduate Research and Creative Activity (CURCA).
Human calcaneal external shape relative to activity and foraging levels
Authors: Harper Christine M. , Ruff Christopher B. , Sylvester Adam D. 
Published: April 2020

The role of the calcaneus in weight‐bearing suggests that it may reflect differences in activity and foraging levels among different modern human populations. While lower limb cortical and trabecular bone have been demonstrated to vary with regard to human behavior, human calcaneal variation has not been studied in this context. Here we analyze the entire calcaneal external shape of recent industrialized modern humans (n=60), Native American agriculturalists (n=47), a transitional Native American population that had not fully adopted agriculture (n=10), and Late Stone Age Khoesan hunter‐gatherers (n=13).Calcanei were either surface or micro‐CT scanned and their external shape analyzed using a three‐dimensional geometric morphometric sliding semilandmark analysis. Semilandmarks were allowed to slide relative to an updated Procrustes average to minimize the bending energy of the thin plate spline interpolation function. Final landmark configurations were aligned using a Generalized Procrustes Analysis. Shape variation was summarized using a principal components analysis. Procrustes distances between different human populations were calculated, and resampling statistics run to test for significant differences.All human populations demonstrate statistically different calcaneal morphologies (p<0.005 for all comparisons). Industrialized and non‐industrialized populations separate from each other across the first six principal components (52.34% of variance). Non‐industrialized populations demonstrate a mediolaterally wider and superoinferiorly taller posterior calcaneus and tuber, as well as a larger peroneal trochlea. This greater robusticity is likely adapted for the increased load through the calcaneus associated with a non‐sedentary lifestyle. The industrialized sedentary modern humans, however, possess a relatively larger lateral plantar process, potentially as a compensatory mechanism for bipedal behaviors in a generally less robust and more antero‐posteriorly elongate calcaneus.Support or Funding InformationThis project was supported by NSF grant # BCS ‐ 1824630.
Flux Of Glucose Through Pentose Phosphate Pathway And Glycolysis In Mouse Hearts Subjected To Low‐Flow Ischemia
Authors: Fan Wei , Lu Daning , Shi Xinwen , He Huamei 
Published: April 2020

IntroductionThe molecular events that lead to irreversible myocardial damage during severe ischemia remain poorly understood. Changes in cytosolic redox status and ability of mitochondria to oxidatively metabolize fatty acids and carbohydrates have been implicated in ischemic Injury. The pentose phosphate pathway (PPP) produces NADPH, which can be used to maintain glutathione in its reduced state, providing protection against oxidative damage. The rate of glycolysis during ischemia determines extent of ischemic injury. We investigated flux of glucose through PPP and glycolysis in mouse hearts subjected to low‐flow ischemia in this study.MethodsIsolated mouse hearts were perfused in Langendorff mode with 10mM [1,2‐13C2]‐glucose for 60min, followed by 90min low‐flow ischemia (perfused at 10% of baseline flow rate). The cardiac function was recorded with PowerLab Data Acquisition Systems. Lipid peroxidation was determined with Colorimetric/Fluorometric Assay Kit. The flux of glucose through the PPP and glycolysis was assessed by the contribution of the doublet (D23, [2,3‐13C2]‐lactate) and singlet (S3, [3‐13C]‐lactate) to the total 13C‐NMR signal of lactate C3 in effluent, respectively.Results90min low‐flow ischemia resulted in both systolic dysfunction (as indicated by decreased left ventricular systolic pressure, rate of tension development and developed pressure) and diastolic dysfunction (as indicated by increased end diastolic pressure and decreased rate of relaxation) accompanied with increased lipid peroxidation. Furthermore, the flux of glucose through PPP increased substantially and correlatively with that through glycolysis.Conclusion[1,2‐13C2]‐glucose can be used to simultaneously assess flux of glucose through PPP and glycolysis. The flux of glucose through both PPP and glycolysis increased during myocardial low‐flow ischemia.Support or Funding InformationThis study was supported by Department Fund.