bluuebunny/crossref_metadata_2025_split huggingface dataset:
A PRIME Labeling Approach For Investigating Dopamine Transporter Endocytic Trafficking In Dopaminergic Cell Lines And Neurons
Authors: Wu Sijia , Uttamapinant Chayasith , Ting Alice , Melikian Haley 
Published: November 2023

The presynaptic dopamine transporter (DAT) facilitates synaptic DA reuptake that spatially and temporally constrains DA neurotransmission. DAT is inhibited by addictive psychostimulants, and the rewarding properties of those psychostimulants require their direct binding to DAT. More recently, aberrant DAT function has been identified in Attention Deficit/Hyperactivity Disorder, Autism Spectrum Disorder and Infantile Parkinsonism, emphasizing the critical role DAT plays in maintaining normal DA tone. DAT robustly traffics to and from the cell surface via the endocytic pathway. Numerous stimuli, including protein kinase C (PKC) activation and amphetamine (AMPH) exposure acutely decrease DAT surface levels in dopaminergic terminals by promoting DAT endocytosis. However, DAT's post‐endocytic fate is unclear and conflicting studies report that DAT targets to either recycling or degradative endocytic compartments, or that DAT may not traffic in bona fide DAergic neurons. These discrepancies may stem from the various approaches used examine DAT trafficking, which rely primarily on either antibody or inhibitory ligand‐based methods that could potentially mistarget internalized DAT. In the current study, we take advantage of a novel PRIME (Probe Incorporation Mediated by Enzyme) labeling strategy to track the DAT endocytic itinerary under basal and stimulated conditions. This approach covalently couples fluorophore to surface proteins that encode a ligase acceptor peptide (LAP), thereby facilitating direct protein visualization without the need for inhibitory ligands or bulky antibodies. We engineered the LAP peptide into the DAT 2nd extracellular loop (LAP‐DAT) and observed highly specific fluorophore labeling at the cell surface in cells expressing LAP‐DAT, but not in cells expressing wildtype DAT. LAP‐DAT expresses, functions, and exhibits PKC‐mediated downregulation comparable to wildtype DAT. Moreover, LAP‐DAT robustly internalized in the immortalized mesencephalic cell line, AN27, allowing us to generate a dynamic flow profile that tracks DAT's movement through the endocytic pathway. Ongoing studies using AAV2‐expressed LAP‐DAT in the striatum of TH‐Cre mice, will permit a rigorous examination of DAT endocytic trafficking in bona fide native dopaminergic neurons in ex vivo tissue samples.Support or Funding InformationNIH R01DA035224 to H.E.M.
Hydrogen Sulfide Inhibits Store‐Operated Calcium Entry by Targeting Orai3
Authors: White Carl , Kipp Jingjing , Fresquez Adriana 
Published: November 2023

Store‐operated Ca2+ entry (SOCE) is activated in response to depletion of endoplasmic reticulum (ER) Ca2+ stores. The molecular machinery consists of the ER‐localized stromal interaction molecule (STIM), which upon store depletion, binds to and opens the plasma membrane channel Orai. Activation of SOCE is subject to redox modifications of both STIM and Orai. The gaseous signaling molecule hydrogen sulfide (H2S) is a known redox modifier of ion channels, however, its potential to affect SOCE has not been explored. To study this we generated HEK293 cells stably overexpressing human STIM1 and then transiently expressed either human Orai1, 2 or 3. The STIM1 and Orai proteins were tagged with YFP and CFP respectively to identify co‐transfected cells and SOCE quantified by Ca2+ imaging. Treatment with the H2S donor GYY4137 (500 μM) had no effect on SOCE mediated by Orai1 or 2 but inhibited the SOCE mediated by Orai3. It is established that H2S functionally affects proteins by modifying the thiol groups of reactive cysteine residues. Sequence analysis of human Orai1, 2 and 3 revealed four cysteine residues in the human and mouse Orai3 protein. Two of these (C101 and C118) are conserved in the Orai1 and Orai2 sequence; however, C226 and C232 are unique to Orai3. To assess there role in mediating H2S‐dependent inhibition these residues were mutated to serine. The SOCE generated by Orai3 C226S and C232S mutants was not significantly different from Orai3 wild type; however, the inhibitory effect of H2S was lost. These data are consistent with a model in which H2S inhibits SOCE mediated only by STIM1/Orai3 and not STIM1/Orai1 or STIM1/Orai2 by targeting specific cysteine residues in Orai3.Support or Funding InformationDePaul‐RFUMS Collaborative Research Grant.
Regulation of Slack KNa Channel Trafficking by p38 MAP Kinase
Authors: Gururaj Sushmitha , Bhattacharjee Arin , Fleites John 
Published: November 2023

Nociceptive sensitization is a central component of neuropathic pain, involving an alteration in the membrane properties of the sensory neurons of the Dorsal Root Ganglion (DRG) such that they have a lowered threshold for activation and hence, increased spontaneous activity. This DRG hyperexcitability manifests as the painful physiological symptoms of hyperalgesia and allodynia in patients of neuropathic pain, in whom there is as yet a large unmet need for medical treatment due to a lack of proper understanding of the precise molecular mechanisms involved. The key role of sodium activated potassium (KNa ) channels in the maintenance of firing accommodation in DRG neurons makes them important targets to study in the context of DRG excitability, which then deems mechanisms of KNa channel regulation as potential ways to regulate DRG hyperexcitability. In this context, we examined the KNa channel Slack's extensive intracellular C‐terminus for possible sites of regulatory protein interactions and found that it contains two putative p38 MAPK phosphorylation sites that are highly conserved across species. While there is no direct link between p38 MAPK and KNa channels, there is evidence for p38 MAPK's role in facilitating neuronal regeneration after nerve crush of the sciatic nerve, indicating that the kinase could be an important player in neuropathy and potentially, in nociceptive sensitization. To answer the question of whether p38 MAPK regulates Slack channels via phosphorylation of the C‐terminus, we have performed electrophysiology experiments to demonstrate that Slack current is subject to modulation by p38 MAPK, and biochemical experiments to show that Slack is basally phosphorylated by p38 MAPK. Site‐directed mutagenesis of the p38 MAPK phosphorylation sites indicates that modulation occurs via phosphorylation at both the sites. Finally, biotinylation assays suggest that the mechanism of modulation of Slack current by p38 MAPK is likely at the membrane via a trafficking mechanism. Together, these results are the first direct demonstration of Slack as a p38 MAPK substrate, and provide evidence for p38 phosphorylation playing a role in Slack channel trafficking and hence, DRG membrane excitability.Support or Funding InformationThis work is supported by Grant NS078184 from the National Institute of Health.
Halocline Behavior and Salinity Preference in the Estuarine Teleost Fish, the Mummichog (Fundulus heteroclitus)
Authors: Marshall William Smithson , Tait Janet C , Mercer Evan W 
Published: November 2023

Mummichogs prefer seawater but have wide ability to acclimate to extreme temperatures and salinities and spawn in dilute brackish water. Haloclines are common features of estuaries, but how fish react to them is not known. The objective was to reveal how mummichogs select microhabitats in the estuary and whether haloclines were a major feature affecting behavior. In the field, minnow trapping revealed that mummichogs move progressively into low salinity warmer water during early spring after ice melt and show significant aversion to colder temperatures and high salinity. First appearance in estuarine shallows occurred above 10 ºC and catch increased to 21 ºC over four weeks. Threespine stickleback (Gasterosteus aculeatus) also preferred warmer low salinity locations, but preferred slow moving streams, whereas mummichogs preferred tidal ponds. In the laboratory, artificial haloclines were made to test isothermal salinity preference, between 28 ‰ full strength seawater (SW, below) and 10 % SW (3.0 ‰, above). Mummichogs of both sexes acclimated to 5 ºC in SW strongly preferred SW. Surprisingly, freshwater (0 % SW) acclimated mummichogs at 21 ºC also preferred SW. In sexually mature fish acclimated to 21 ºC SW, only the males preferred SW; the females showed no significant preference for SW, meaning they freely entered low salinity. SW preference was manifested by a stereotypic passive aversion to the dilute upper layer at the halocline. We conclude that the overall movement of mummichogs into summer breeding grounds of low salinity is driven by maturation of females and their preference for warmer water (regardless of salinity) that overcomes the general tendency of the immature and male fish to avoid dilute waters.Support or Funding InformationSupported by NSERC Discovery Grant RGPIN3698‐2009 to WSM and UCR scholarship to JCT.
Chemosensory Prey Preference in Neotropical Gastropod‐eating Snakes (Dipsadinae: Dipsadini)
Authors: Sheehy Coleman M. , Tovar Ruben U. 
Published: November 2023

Neotropical snakes in the tribe Dipsadini are gastropod specialists, and many possess a suite of morphological and behavioral characteristics generally accepted as adaptations for extracting land snails from their shells. To test the assumption that these snakes prefer gastropod prey, we collected three Dipsas gaigeae, six Tropidodipsas philippii, two T. annuliferus and three Sibon nebulatus and offered to them the scents of four prey items (i.e., snail, earthworm, cricket and frog) plus two controls. Scents were offered via a cotton‐tipped swab, and the response variable was the number of tongue flicks directed towards the swab in 60s. Mean maximum tongue‐flick responses for each species were analyzed using ANOVA and rmANOVA. Dipsas gaigeae, S. nebulatus and T. annuliferus preferred gastropod prey to other potentially available prey, whereas T. philippii showed a strong preference for both gastropods and earthworms. Unlike the other species, T. phillippi consumed live earthworms, suggesting that this species likely eats earthworms in nature in addition to gastropods. These results suggest that earthworms may be the ancestral diet of the Dipsadini and highlight the importance of testing dietary assumptions in order to better understand the evolution of dietary specialization.Support or Funding InformationWe thank J. Reyes‐Velasco, C. Cox, J. Streicher, G. Rivas, T. Barros and M. Ingrasci for assistance with fieldwork and acquiring live snakes. A. Pires da Silva kindly provided space for conducting this study in the animal room facility at the University of Texas at Arlington. We thank O. Flores‐Villela, C. Franklin, A. (Beto) Mendoza and A. Monzón for their assistance with obtaining permits. This study was supported by NSF grant DEB 0613802 to J. Campbell and O. Flores‐Villela. The use of live snakes for this study fully complied with approved UTA Institutional Animal Care and Use Committee (IACUC) protocol A07.032.
Mutations in O‐GlcNAc Transferase Linked to X‐linked Intellectual Disability
Authors: Wells Lance 
Published: November 2023

O‐GlcNAc is a regulatory post‐translational modification of serine and threonine residues of nuclear and cytoplasmic proteins that has been implicated in multiple biological processes including epigenetics. In humans, single genes encode enzymes for its attachment (O‐GlcNAc transferase (OGT)) and removal [O‐GlcNAcase (OGA)]. An X‐chromosome exome screen identified a missense mutation in the tetratricopeptide (TPR) domain of OGT (762G>T (p.L254F)) that segregates with X‐linked intellectual disability (XLID) in an affected family. A decrease in steady‐state OGT protein levels, in part due to a shorter half‐life of L254‐OGT, was observed, as predicted by in‐silico modeling, in isolated lymphoblastoid cell lines from affected individuals compared to controls. Recombinant expression of L254‐OGT demonstrated that the enzyme is active against in vivo protein substrates and in vitro peptide substrates. Surprisingly, steady‐state global O‐GlcNAc levels and the O‐GlcNAcome of the lymphoblasts remain grossly unaltered. Affected lymphoblastoids display a marked decrease in steady‐state OGA protein and mRNA levels, implying that a compensation mechanism exists, albeit imperfect given the phenotype of the patients, for maintaining global O‐GlcNAc levels. L254F‐OGT samples also display decreased luciferase reporter expression driven from the proximal promoter of OGA, suggesting that OGT regulates the gene expression of OGA. We observed an enrichment of the OGT containing transcriptional repressor complex mSin3A‐HDAC1 at the proximal promoter region of OGA in affected individuals, which would explain the decrease in OGA gene expression with reduced OGT protein levels. Global transcriptome analysis of L254F‐OGT lymphoblastoids compared to controls revealed a small subset of genes, many involved in chromatin remodeling, that are differentially expressed in affected patients. Further investigation has revealed another XLID family with the SNP E338G in OGT segregating with XLID and thus we have uncovered the first human disorder associated with mutations in OGT. Given that both mutations are in the TPR domains on OGT, we are currently exploring defining the OGT interactome.Support or Funding InformationThis work was supported in part by a medical research grant from the W.M. Keck Foundation and by the National Center for Biomedical Glycomics (P41GM103490).
Synergistic role of hemin in endothelial cell activation in the lungs of sickle cell mice
Authors: Hu Xingang , Zheng Zhen , Alexeyev Mikhail F , Dickerson Carol , Meiler Steffen E , Wu Songwei 
Published: November 2023

Although sickle cell disease (SCD) has long been characterized by typical clinical picture of sickle hemoglobin, chronic hemolytic anemia, and recurring episodic vaso‐occlusive crisis, mechanistic cascades linking hemolysis to vaso‐occlusion still remain largely unclear. Notably, the vascular endothelium in patients with SCD exhibits a consecutively activated, pro‐inflammatory phenotype even under steady state conditions, and a further enhanced inflammatory response occurs during vaso‐occlusive crisis. Recent studies have suggested that the hemolytic products, including heme and its oxidized form hemin, may directly target the endothelium, promote endothelial cell pro‐inflammatory activation, and consequently trigger vaso‐occlusion. Thus, the present study examined a potential mechanism of hemin in promoting pro‐inflammatory lung endothelial cell activation in SCD. Fluorescence microscopy was first performed in cultured rat pulmonary microvascular endothelial cells (PMVECs) that were stably transduced with a GFP‐tagged P‐selectin to examine the dynamics of P‐selectin translocation following stimulation with hemin (400 nmol/L). Indeed, hemin markedly stimulated P‐selectin translocation from cytosol to the plasma membrane leading to P‐selectin surface expression. Such hemin‐induced P‐selectin surface expression was nearly abolished by the membrane‐permeable radical scavenger tempol (20 μmol/L), indicating that the stimulative effect of hemin is mediated by cell oxidative stress. Next, an ex vivo polymorphonuclear neutrophil (PMN)‐perfused lung model was employed to determine whether hemin induces potentiated intracapillary neutrophil retention in SCD lungs. Lungs isolated from 12‐week‐old transgenic humanized homozygous SCD (SCD−/−) and heterozygous (SCD+/−) mice were assessed for intravascular PMN sequestration using immunohistochemistry for leukocyte myeloperoxidase following hemin perfusion (10 μmol/L, 15 min) followed by perfusion of non‐stimulated rat PMNs (1.0 × 105/mL, 20 min). Hemin‐stimulated intracapillary PMN sequestration was observed to be more pronounced in SCD−/− lungs as compared to SCD+/− lungs. The potentiated hemin‐induced PMN sequestration in SCD−/− lungs was dramatically attenuated when the lungs were perfused with a P‐selectin antibody (0.5 mg per lung set, 5 min), tempol (150 μmol/L, 5min), or, to a lesser degree, endothelin receptor A (ETA) antagonist BQ‐123 (1 μmol/L, 5 min), but not endothelin receptor B (ETB) antagonist BQ‐788 (1 μmol/L, 5 min), prior to hemin perfusion. Taken together, these observations suggest that hemin, by activating P‐selectin surface expression via intracellular reactive oxygen species generation, acts additively or synergistically to promote alveolar capillary endothelial pro‐inflammatory phenotype in SCD lungs.Support or Funding InformationHL‐117684 (to S. Meiler) and HL‐066299 (to S. Wu)
Reduction in Edema Cuff Size is not the Mechanism of Lung Protection Afforded by Airway Pressure Release Ventilation (APRV)
Authors: Jain Sumeet Vidya , Kollisch‐Singule Michaela , Roy Shreyas , Pasquale Gabriella , Sweeney Tara , Satalin Josh , Andrews Penny , Habashi Nader , Nieman Gary , Gatto Louis A 
Published: November 2023

IntroductionWe have shown that preemptive application of APRV significantly reduces the incidence of acute respiratory distress syndrome (ARDS). The exact protective mechanism is currently not known.ObjectiveTo determine if the mechanism of this protection was a reduction in the size of pulmonary vascular edema cuffing.Methods14 swine were randomized into three groups: naïve (n=5), non‐protective ventilation (NPV; n=5), and airway pressure release ventilation (APRV; n=4). Naïve pigs did not receive lung injury. A fecal clot and gut ischemia/reperfusion‐induced ARDS model was used in both the NPV and APRV groups. Following injury, both groups were managed on their respective ventilator settings and sacrificed after 48 hours. Lung tissue was fixed and histology examined at 10× and 40× magnification. Lumen, tunica media, tunica adventitia, and lymphatics were identified, outlined, colored, and then quantified (Figure). The following parameters were further evaluated for each vessel: lumen area, lumen roundness, tunica media area, tunica adventitia area, and lymphatic area. Clinical parameters and histologic lung injury scoring were analyzed.ResultsVessel lumen roundness and tunica media area did not vary between groups (p>0.05). Lumen area was smaller in the injury groups (NPV/APRV) than in the naïve animals (p<0.05). The perivascular cuff, i.e. tunica adventitia and lymphatic vessels, was larger in the injured animals compared with naïve (p<0.05), without difference between ventilatory strategy. The tunica adventitia occupied a larger percent of total vessel area in the injured animals; however, lymphatics did not occupy a larger percent of the cuff compared with naïve, correlating instead with vessel size. Naïve pigs had lower wet/dry ratio compared with NPV pigs (5.47 vs. 8.82; p<0.05) without difference between Naïve and APRV groups (5.47 vs. 7.11, p>0.05) or APRV and NPV groups (7.11 vs 8.82; p>0.05). PaO2/FiO2 ratio was higher in the APRV vs. NPV group (p<0.05), and histologic scoring showed decreased lung injury.ConclusionDespite increased lung protection using APRV, there was no difference in edema cuff size. Therefore, changes in edema cuff size do not contribute to APRV‐induced lung protection.
The Effect of Obese Levels of Leptin on Peripheral Chemoreception
Authors: Pye Richard L. , Roy Arijit , Wilson Richard J.A. , Wyatt Christopher N. 
Published: November 2023

Obesity rates are increasing rapidly worldwide. Breathing and cardiovascular systems’ function are directly impacted by obesity and contribute to obesity related pathology. Leptin receptors are present in the oxygen sensing type I cells of the carotid body and in carotid sinus nerve afferent fibers. The adipokine leptin is a respiratory stimulant with known central effects. We have previously reported that acute administration of obese levels of leptin causes a measurable and long lasting change in Ca2+ signal in isolated carotid body type I cells as well as augmenting BKCa channel activity. Here we report that 100nM wortmannin, a PI3K inhibitor, blocks leptin induced Ca2+ signaling in these cells. Furthermore we show that in an ex‐vivo arterially perfused carotid body preparation that leptin causes a dose dependent increase in baseline firing rates under normoxia and an increase in the size of the sensory response to hypoxic challenge that does not wash out. These data show that leptin alters carotid body function acutely and suggest that studying carotid body function in chronic models of obesity is warranted.Support or Funding InformationSupported by NIH R01‐HL091836
Activation of a Non‐Selective Cation Channel by Store‐Operated and Receptor‐Mediated Elevation of [Ca2+]i in Rat Carotid Body Glomus Cells
Authors: Kim Donghee , Wang Jiaju , Hogan James O 
Published: November 2023

Our recent studies show that a non‐selective cation channel (NSC) is highly expressed in rat glomus cells. An increase in Ca2+ influx via the voltage‐dependent Ca2+ channel (VDCC) caused by depolarizing stimuli such as hypoxia, low pHo and high [KCl]o activated NSC. The effect of [Ca2+]i rise by mechanisms other than via VDCC on NSC activity is not yet known. Here we studied the role of store‐operated Ca2+ entry (SOCE) and receptor‐mediated [Ca2+]i rise on NSC activity. Patch‐clamp single channel recording and fura‐2 fluorescence signal were used. SOCE was produced by incubating cells in Ca2+‐free solution containing 1 uM thapsigargin and then adding Ca2+ back to the solution. The rapid rise in [Ca2+]i associated with SOCE caused a strong activation of NSC. Receptor‐mediated [Ca2+]i rise was produced using Angiotensin II (100 nM) and muscarine (10 uM), which stimulate the Gq‐PLC pathway and elevates [Ca2+]i in glomus cells. Both Ang II and muscarine produced a significant activation of NSC. Caffeine that causes release of Ca2+ from the endoplasimc reticulum also elevated [Ca2+]i and activated NSC. GABA, dopamine, endothelin‐1, 5‐HT did not elevate [Ca2+]i and did not activate NSC. Adenosine produced a weak elevation of [Ca2+]i but did not activate NSC. Our results indicate that a stimulus that produces an increase in [Ca2+]i, regardless of which signaling pathway (VDCC, SOCE and receptor‐mediated) is used, can activate NSC to help depolarize glomus cells.Support or Funding InformationSupported by NIH grant