bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cellular and Proteomic Characterization of the Innate Immune Response in Bat Stars (Patiria miniata)
Authors: Andrus Avery Kay , Livingston Brian Thomas 
Published: November 2023

Bat star populations on the west coast have been declining due to the Sea Star Wasting Syndrome. This tissue‐decaying epidemic involves a viral infection and skin lesions leading to disintegration in sea stars. The mechanisms causing decay in the symptomatic individuals are still unknown. We considered the immune response in bat stars after the appearance of skin lesions to determine how immune cells react and which proteins are expressed throughout the viral infection and tissue disintegration. We hypothesized that the viral infection is met with an increase in immune cell concentration and phagocytic activity, and an increase in the secretion of immune‐ and stress‐response related proteins into the coelomic fluid in activated defensive pathways. The morphology, behavior, and concentration of the immune cells per mL of coelomic fluid in healthy and wasting bat stars were compared by extracting coelomic fluid and staining nuclei, photographing the cells, and counting them. Immune cells were extracted at healthy and middle time points with no visible lesions, and at an endpoint once visible lesions were present; the cells were counted and incubated with fluorescent bacteria. Phagocytes, cells that engulf invading pathogenic cells, were found in higher cell concentrations after viral infection and the appearance of lesions, showing higher levels of activity and consuming more fluorescent bacteria. The reactions of the cells to these pathogens were observed in coelomic fluid from healthy and wasting sea stars. From the coelomic fluid samples taken throughout the progression of the disease in several individual sea stars, LC/MS/MS analysis and NCBI BLAST characterization of the proteins present showed dynamic abundance patterns in secreted proteins related to cell signaling, stress, and immune response. We are attempting a more thorough characterization of the proteins involved in the immune reaction to better define the defense mechanisms occurring in the bat stars during the response to the viral challenge. The changes in abundance of certain proteins may give clues about the patterns of disintegration occurring at the cellular level and the pathways involved in the tissue disintegration. We may also be able to identify novel antimicrobial or anti‐viral molecules of pathways, with potential for future use in tissue preservation.Support or Funding InformationCOAST Grant, CSULB 2015 Graduate Summer Research Fellowship.
Abuse Liability and Anti‐Addiction Potential of the Atypical Mu Opioid Receptor Agonist IBNtxA
Authors: Keck Thomas M , Verchio Vincent , Wu Chun , Moura‐Letts Gustavo 
Published: November 2023

OBJECTIVEIBNtxA is a novel μ opioid receptor (MOR) agonist, structurally related to the classical MOR antagonist naltrexone, that preferentially signals through truncated MOR splice variants. This unique pharmacological profile produces potent analgesia with reduced side effects, including no conditioned place preference (CPP) when tested at a single dose. The purpose of this study is to 1) evaluate a range of IBNtxA doses to more fully assess its abuse liability and 2) determine the effects of IBNtxA on morphine CPP expression and reinstatement of morphine CPP.METHODSIBNtxA was synthesized and compared to morphine in standard CPP expression assays. Following morphine CPP training, IBNtxA was tested for its effects in inducing CPP reinstatement on its own or attenuating morphine‐primed reinstatement.RESULTS and CONCLUSIONSIBNtxA is an intriguing preclinical drug that opens the door to new MOR splice variant‐selective pharmacotherapeutics with effective analgesic properties but reduced side effects. IBNtxA may have utility as an adjunct therapy in agonist replacement strategies (e.g., methadone). Current collaborative efforts are aimed at developing novel analogues of IBNtxA for further analysis and understanding ligand‐receptor interactions across MOR splice variants using molecular modeling.Support or Funding InformationRowan University Seed Fund; travel support from Dean of College of Science and Mathematics and the Rowan University Assistant Professor and Instructor Travel Fund.
Differential role of G protein signaling regulator RGS7 on rewarding effects of opioids and psychostimulants
Authors: Sutton Laurie , Ostrovskaya Olga , Dao Maria , Smith Roy , Martemyanov Kirill 
Published: November 2023

The endogenous opioid system is critical for natural reward responses that drive positive behavioral reinforcement, as well as the reinforcing effects associated with addictive substances, such as morphine. The rewarding and reinforcing properties of many drugs of abuse depend on the activation of the m‐opioid receptor (MOR). MOR is a G‐protein coupled receptor whose signaling efficiency is controlled by the regulator of G‐protein signaling (RGS) proteins. In vitro studies have demonstrated that RGS7, a member of the R7 family of RGS proteins, negatively regulates MOR signaling via inhibitory G‐proteins of the Gi/o class. Co‐labeling studies performed in the CNS show that RGS7 and MOR are co‐expressed in the same neuronal population. However, the impact of RGS7 on MOR signaling in vivo is unknown. This study utilizes both genetic and viral approaches to ablate RGS7 both globally and in specific neuronal populations to determine the contribution of RGS7 in reward‐related behaviors. Global RGS7 knockout mice display enhanced morphine‐induced conditional place preference (CPP) and increased locomotor activity in response to morphine. Conditional elimination of RGS7 in striatal neurons results in similar enhancement of morphine‐induced reward in the CPP test. Using an operant self‐administration task these conditional RGS7 knockout mice also displayed increase active lever pressing, as well as a vertically shifted dose‐response profile of morphine‐intake. Together this suggests that RGS7 modulates the reinforcing properties of morphine. To investigate the impact RGS7 has on morphine‐induced alteration in neuronal excitability and plasticity of glutamatergic synapse, electrophysiology approaches were applied to those mice that underwent morphine self‐administration. It was found that RGS7 exerted its effects by controlling morphine‐induced changes in excitability of medium spiny neurons in the nucleus accumbens (NAc) and gating the compositional plasticity of AMPA and NMDA receptors. We further studied the role of RGS7 in reward‐related behaviors in response to other drugs of abuse, such as psychostimulants. Interestingly, mice lacking RGS7 KO exhibit similar responses to cocaine‐induced CPP as their wild‐type littermates. Overall, the results show that RGS7 in the NAc specifically control the rewarding and reinforcing effects of opioids but not of psychostimulants.Support or Funding InformationThis work was supported by the National Institutes of Health Grants DA026405, DA036082, and DA036596 and by the Canadian Institutes of Health Research (CIHR) Fellowship.
The Effect of Ventral Tegmental Area Rictor Knockout on Susceptibility to Chronic Social Defeat Stress and Stress‐induced Changes in Morphine Reward
Authors: Kaska Sophia , Mazei‐Robison Michelle 
Published: November 2023

There is significant co‐morbidity of addiction and depression and this may be due to dysregulation of common brain circuits. For example, similar alterations in signaling in the ventral tegmental area (VTA) have been identified in response to both chronic morphine and chronic social defeat stress (CSDS), a rodent model of depression. We are particularly interested in target of rapamycin complex 2 (TORC2) signaling in the VTA as it has been established to play a critical role in morphine reward and morphine‐induced neuroadaptations. However, whether it plays a similar role in stress‐induced changes in morphine reward, or stress susceptibility itself, is unknown. To address these questions, we used viral and genetic approaches to decrease VTA TORC2 signaling by knocking out the expression of Rictor, a TORC2 component protein necessary for TORC2 function. Floxed‐Rictor mice were either crossed with a TH‐Cre mouse line to eliminate TORC2 signaling in all catecholaminergic neurons or underwent stereotaxic surgery to specifically decrease TORC2 signaling in the VTA via AAV‐Cre infusion. The mice were then subjected to chronic physical or emotional CSDS and their susceptibility to stress was measured using the social interaction (SI) test. Following SI testing, mice underwent a two‐bottle choice assay to assess voluntary morphine preference and consumption. In contrast to previous data that found that decreasing AKT signaling was sufficient to increase susceptibility to stress, we found that decreased VTA TORC2 signaling did not alter stress susceptibility. However, we did observe a difference in morphine consumption, as Rictor knockout mice (both TH‐Cre positive and AAV‐Cre) that underwent stress consumed significantly more morphine than stressed controls (intact TORC2 signaling). Additionally, following stress, Rictor knock‐out mice also exhibited an increase in water and sucrose consumption, suggestive of an overall increase in consummative drive. Importantly, these changes were only evident in mice following stress, suggesting a novel role for VTA TORC2 signaling in stress‐mediated changes in consumption.Support or Funding InformationThis work was supported by NIH T32 GM092715 (SK) and NIDA DA037426 (MMR).
Slick (Kcnt2) sodium‐activated potassium channels limit nociceptor excitability and pain
Authors: Tomasello Danielle Leigh 
Published: November 2023

The physiological functions of the large conductance Slick sodium‐activated potassium (KNa) channels have yet to be identified. Immunolabeling of Slick channels in murine dorsal root ganglia (DRG) and trigeminal ganglia indicated localization exclusively to proinflammatory peptide calcitonin‐gene related peptide (CGRP) containing small and medium sized neurons, indicating a possible function in nociception. Staining was found within the spinal cord and at the nerve endings in the paw suggesting a role in both pain detection and neurotransmitter release. Strikingly, we found Slick channels localized to large dense core vesicles (LDCV) containing CGRP in DRG neurons. We then studied the phenotypic consequences of Slick deletion using a novel Slick‐LacZ reporter knockout (KO) mouse line. First, we tested the electrophysiological properties of peptidergic DRG neurons from KO and WT mice. We found a decrease in the total outward potassium current, a decrease in action potential rheobase, and an increase in the height of action potential from DRG neurons isolated from Slick KO mice compared to wildtype (WT) neurons, indicating that Slick channels regulate nociceptor excitability. Utilizing models for neuropathic and inflammatory pain, we then tested thermal hyperalgesia on KO and WT littermates. Prior to nerve injury, Slick KO mice exhibited sensitized basal nociception reinforcing our electrophysiology data, and following injury KO mice had heightened hyperalgesia compared to WT mice. In the inflammatory model, injection of complete Freund's adjuvant into the hindpaw caused prolonged but reversible thermal hyperalgesia in WT mice. Hyperalgesia was exacerbated in Slick KO mice and did not exhibit recovery during the time course tested suggesting that the recruitment of Slick channels to the membrane is important for pain recovery. We stimulated DRG neurons for CGRP release and found Slick channels contained within CGRP positive LDCV translocated to the neuronal membrane. We then assessed the consequence of Slick deletion after CGRP stimulated release and found Slick KO neurons were more excitable compared to WT neurons in both control and stimulated release. Our data indicate that Slick channels limit the excitability of specifically CGRP peptidergic neurons and reduce pain behavior after injury and inflammation. Given the specificity of Slick expression to CGRP positive neurons only, Slick channels are desirable pharmacologic targets for the treatment of pain and inflammation.Support or Funding InformationThe research was supported by the National Institute of Health, Grant NS078184 (to A.B.). Travel Award to EB 2016 provided by Hello Bio, Bristol UK.
Depression of Home Cage Wheel Running: A Novel Method to Assess Spontaneous Migraine Pain
Authors: Kandasamy Ram , Calsbeek Jonas J , Morgan Michael M 
Published: November 2023

Migraine is the most common neurological disorder in the world, yet current treatments have limited efficacy and repeated administration can cause medication‐overuse headache. Although new treatments are desperately needed, the difficulty of assessing migraine pain in laboratory animals has limited drug development. Most animal studies of migraine assess mechanical allodynia in the face and hindpaw of the rodent. Although useful, this measure does not assess spontaneous, unevoked headache pain – a key characteristic of migraine. The objective of the present study is to test the hypothesis that home cage wheel running is a sensitive and objective method to assess spontaneous migraine pain in the rat – an approach that mimics the decrease in activity used to diagnose migraine in humans. Adult male and female rats were implanted with a cranial cannula aimed at the periosteal space above the dura mater. Rats were allowed to recover for 8 days in their home cage with unlimited access to a running wheel. Migraine pain was induced by microinjecting 10μL of 10% allyl isothiocyanate (AITC) onto the dura. Control rats were injected with mineral oil. Rats were injected with the anti‐migraine agent sumatriptan (1 mg/kg, s.c.) or saline 30 min following the intracranial injection. Administration of AITC into the periosteal space caused a decrease in running in both male and female rats that lasted 4–7 hrs. Administration of sumatriptan prevented this depression of wheel running. The fact that sumatriptan prevented migraine pain‐induced depression of home cage wheel‐running demonstrates that home cage wheel running is a sensitive and reliable measure of spontaneous headache pain in rats.Support or Funding InformationThe study was supported by State of Washington Initiative Measure No. 171 and Washington State University.
A Novel Activator of Canonical Wnt Signaling
Authors: Baruah Jugajyoti , Wary Kishore K , Hitzman Ryan , Zhang Jane 
Published: November 2023

RationaleAs many competitive inhibitors of Glycogen Synthase Kinase‐3 (GSK‐3)β exhibit off‐target effects, we investigated the ability of a newly identified small molecule allosteric inhibitor of GSK‐3β, called Tideglusib (TDG), to activate canonical Wnt signaling.Methods and ResultsThe treatment of cultured endothelial cells (ECs) with Wnt3a or TDG stabilized β‐catenin polypeptide species in a dose‐dependent manner. A time‐course analyses showed increased stabilization of β‐catenin in ECs in response to TDG. The addition of TDG in ECs induced increased expression of not only transcription factor Nanog, but also Cyclin‐D1 and Vascular Endothelial Growth Factor Receptor‐2 (VEGFR‐2) at both protein and mRNA levels. Microscopic analyses of ECs stained with anti‐VE‐cadherin antibody provided evidence of the disappearance of VE‐cadherin from the adherens junctions, while increased accumulation of β‐catenin in the nucleus. Importantly, in a TOP‐Flash Luciferase assay, TDG showed significant activation of Wnt pathway in both ECs and 293T cells, also accompanied by increased nuclear β‐catenin. Pretreatment of ECs with TDG followed by Propidium iodide (PI) and Annexin‐V labeling showed increased survival and decreased cell death (p<0.05 vs control groups). Importantly, TDG treatment significantly increased the incorporation of 5‐bromo‐2′‐deoxyuridine (BrdU) in ECs. In addition, in a scratch assay and Boyden‐chamber migration experiments indicated the ability of TDG to induce migration of ECs. Furthermore, in an aortic ring assay TDG induced robust neovessel formation.SummaryOur results demonstrate that an allosteric inhibition of GSK‐3β stabilizes β‐catenin, thereby upregulating Cyclin‐D1, Nanog, and VEGFR‐2 that are critical regulators of cell proliferation and stem cell self‐renewal.Support or Funding InformationAmerican Heart Association (AHA) Mid‐west affiliate Predoctoral Fellowship 2015–2016
Optimizing the analysis of bias to produce more accurate results
Authors: Stahl Edward , Ehlert Frederick J , Bohn Laura M 
Published: November 2023

Seven transmembrane receptors interact with a number of intracellular targets including multiple heterotrimeric G proteins, G protein receptor kinases, and the scaffolding protein β‐arrestin2. For more than a decade, a body of evidence has supported the presence of specific ligand‐receptor complexes that can preferentially stimulate a subset of intracellular targets. This has led to screening and development of ligands, for a number of different receptors, that demonstrate preferential signaling. An example of this is the discovery of the kappa opioid receptor (KOR) agonist 6′‐guanidinonaltrindole (6′‐GNTI). This agonist stabilizes an active receptor state of the receptor that prefers to stimulate G protein activity over the recruitment of β‐arrestin2. We have recently published a novel, competitive, model that was well suited to analyze the bias of 6′‐GNTI.In the studies presented here, we demonstrate a Schild‐type model we have constructed to quantify ligand bias. This method is based on the type of experimental design commonly represented in a Schild plot. Using experimental evidence from the KOR and 6′‐GNTI, as well as data from Monte Carlo simulations, we compare the Schild‐type model with two previously defined models: the standard model and our competitive model. We demonstrate that the Schild‐type model produces a reduction in the error of the parameter estimates when analyzing both the simulated and experimental data (compared to the error produced using the standard model). We are able conclude that, although the cost of employing the Schild‐type model or the competitive model may be greater than for the standard model, these models provide the benefit of producing more accurate and precise bias estimates. From these findings we then introduce a spectrum of rigor for evaluating the bias of competitive ligands.Support or Funding InformationThis work is supported by a grant from the National Institute on Drug Abuse (R01DA033073 LMB).
Characterization of Novel Biased KOR Agonist‐Mediated Cell Signaling
Authors: Ho Jo‐Hao , Lovell Kimberly , Zhou Lei , Frankowski Kevin , Aubé Jeffrey , Bohn Laura 
Published: November 2023

Kappa opioid receptors (KORs) are widely expressed in the central nervous system and are involved in the regulation of pain perception. Therefore, KORs have been attractive drug targets for treating pain; however, KOR agonists also produce sedation and dysphoria. Recent evidence suggests that while G protein signaling may promote KOR‐mediated antinociception, βarrestin‐mediated signaling may lead to the dysphoric effects. In an effort to begin to determine whether KOR activation without βarrestin2 recruitment would maintain the therapeutic potency of KOR agonists while avoiding the aversive effects, we evaluated two classes of KOR agonists, triazole analogue and isoquinolinone analogue, both of which potently activate G protein coupling but weakly recruit βarrestin2. Canonically, KOR couples to inhibitory G proteins that suppress adenylyl cyclase. However, the biased KOR agonists have very weak potency for inhibiting adenylyl cyclase in cell‐based signaling assays. Therefore, we hypothesize that these novel KOR agonists preferentially transduce signaling through different types of Gα or Gβγ proteins. In this study, we compare these novel KOR agonists to the reference compound, U69,593, for their abilities to activate three different KOR‐mediated G protein signaling events: adenylyl cyclase activity by cAMP HTRF technology, G protein‐coupled inwardly‐rectifying potassium (GIRK) channel activity by FLIPR potassium technology, and intracellular calcium mobilization by FLIPR calcium technology. These novel KOR agonists display weak potency in regulating adenylyl cyclase activity and stimulating calcium response; however, they potently activate GIRK channels in cell‐based assays. Together, these results suggest that the novel KOR agonists display different signatures in KOR‐mediated G protein signaling. This study provides insights into the novel KOR agonist‐mediated signaling pathways which will inform future behavioral studies.Support or Funding InformationThis work was supported by NIH/NIDA R01 DA031927 to LMB and JA.
Pharmacological Properties of Chromobacterium violaceum Violacein at the Human Serotonin 2C Receptor
Authors: Fears Letimicia , Curtis Mary , Johnson Terrance , Fentress Hugh 
Published: November 2023

Serotonin (5‐HT), a neurotransmitter, plays a role in many physiological responses by binding to 16 receptor subtypes. The 5‐HT2C receptor subtype is a 7‐transmembrane, G protein‐coupled receptor (GPCR) that is involved in neuronal excitability, spatial learning, mood, and appetite. The microorganism Chromobacterium violaceum produces a purple pigment, violacein, which can be extracted from liquid cultures. Violacein has antibiotic, antileishmanial, antiviral and antitumoral effects in various cancer cell lines. Violacein is similar in structure to 5‐HT, however, no one has ever reported the activity of violacein at 5‐HT receptors. To investigate the effect of violacein on 5‐HT2C receptor trafficking, Human Embryonic Kidney (HEK) 293 cells expressing fluorescently‐tagged 5‐HT2C receptor were treated with 5‐HT, violacein, water or vehicle, fixed and visualized with fluorescent microscopy. Violacein treatment did not cause receptor internalization. Given our recent discovery that the 5‐HT2C receptor can activate the JAK/STAT pathway, we examined if violacein could impact this activation. HEK 293 cells expressing 5‐HT2C receptor were treated with violacein or pretreated with violacein followed by incubation with 5‐HT. Phosphorylation states of JAK2 and STAT3 were examined by immunoblotting. Preliminary data suggests that violacein may hinder activation of STAT3 but further study is required. We will also investigate the effect of violacein on G‐protein activation by measuring phosphoinositide hydrolysis. Affinity for violacein to the 5‐HT2Creceptor will be investigated by competition binding.Support or Funding InformationDepartment of Education Title III HBGI Grant No. P031B141010