bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lorcaserin decreases the reinforcing effects of 3,4‐methylenedioxypyrovalerone (MDPV) and cocaine in Sprague‐Dawley rats
Authors: Gannon Brenda M , Rice Kenner C , Collins Gregory T 
Published: November 2023

Lorcaserin is a serotonin (5‐HT) 2C receptor‐preferring agonist that is currently approved by the Food and Drug Administration to treat obesity; however, its behavioral effects are also mediated by 5‐HT 2A and 1A receptors. Agonists acting at 5‐HT2C receptors are known to dampen dopamine neurotransmission, an effect that could be beneficial for treating drug abuse. 3,4‐Methylenedioxypyrovalerone (MDPV) is a cocaine‐like monoamine reuptake inhibitor that is commonly found in abused bath salt preparations. Recent studies have demonstrated lorcaserin decreases the reinforcing effects of cocaine in rats and monkeys, and the present study aimed to: (1) determine whether lorcaserin was equally effective at decreasing the reinforcing effects of MDPV and cocaine; and (2) determine the receptor(s) responsible for the effects of lorcaserin on cocaine and MDPV self‐administration. Male Sprague‐Dawley rats (n=6) were injected with lorcaserin (0.1–5.6 mg/kg, IP) 25 minutes before the initiation of MDPV or cocaine self‐administration sessions. Lorcaserin was equipotent at suppressing responding for MDPV and cocaine; however, individual differences in sensitivity to lorcaserin were observed. To determine which receptor mediated the effects of lorcaserin, rats were administered 5‐HT1A (WAY100635, 0.178 mg/kg), 5‐HT2A (MDL100907, 0.1 mg/kg), or 5‐HT2C (SB242084, 0.1 mg/kg) receptor antagonists (IP) 15 minutes prior to lorcaserin treatment. Antagonism of 5‐HT2C (but not 5‐HT1A or 5‐HT2A) receptors reversed the effects of lorcaserin on MDPV and cocaine self‐administration. Taken together, these data indicate lorcaserin may be an effective pharmacotherapy against MDPV and cocaine abuse through its agonist actions at 5‐HT2C receptors.Support or Funding InformationThis research was supported by the National Institutes of Health (NIH) and the National Institute on Drug Abuse (NIDA) R01DA039146 (GTC) and T32DA031115 and the NIH Intramural Research Programs of the NIDA and the National Institute of Alcohol Abuse and Alcoholism (NIAAA) (KCR).
Lessons Learned from PKA: from Motifs to the Dynamic Assembly of Isoform‐specific Macromolecular Switches
Authors: Taylor Susan S. 
Published: November 2023

Twenty‐five years after the first protein kinase structure, we are still learning about the fundamental properties that define this large enzyme family, and cAMP‐dependent protein kinase (PKA) continues to serve as a prototype for the superfamily. Each protein kinase has evolved to be a highly regulated and dynamic molecular switch. For most kinases the assembly of the active kinase is dynamically and transiently regulated by the alignment of a hydrophobic regulatory spine. In the case of PKA, however, the active catalytic (C) subunit is then packaged as an inactive R2C2 holoenzyme with dimeric regulatory (R) subunits that are receptors for cAMP. PKA thus combines two of the major regulatory mechanisms in biology, protein phosphorylation and cAMP signaling. Specificity in PKA signaling is achieved in large part by four functionally non‐redundant R‐subunits (RIa, RIb, RIIa, and RIIb), and targeting of these holoenzymes to specific sites in the cell creates yet another layer of specificity. The functional non‐redundancy of the R‐subunits is reflected in the structural diversity of the holoenzymes. Many diseases result from dysfunctional PKA signaling, and these diseases also reflect the diversity and unique functions of the R‐subunit isoforms. While we have learned much from the individual subunits, it was not until we solved the structure of the full length R2C2 holoenzyme that we could appreciate allostery and also why the four holoenzymes were functionally so different. Although each R‐subunit has the same domain organization that includes an N‐terminal dimerization/docking (D/D) domain and two tandem cAMP binding (CNB) domains, the quaternary organization of each holoenzyme is distinct due in large part to the intrinsically disordered linker that joins the D/D and CNB domains. Embedded in the linker is an inhibitor site that docks into the active site of the C‐subunit in the holoenzyme rendering it inactive, and these linkers in large part drive the conformational state of each holoenzyme. Our major challenge now is to understand how the flexible linkers drive the assembly and regulation of the holoenzyme. Capturing this dynamic space is a major challenge for PKA signaling and indeed a fundamental challenge for all protein kinases. Recognizing this critical roadblock, we are now using cryo Electron Microscopy (cryoEM) in parallel with crystallography and SAXS/SANS to explore the domain dynamics that contribute to PKA signaling.Support or Funding InformationFunded in part by NIH grants GM34921 and DK54441.
Interactions between the G Protein‐coupled Estrogen Receptor and the Stromal Interaction Molecule 1
Authors: Tran Kim , Terry Lara E , Gebert‐Oberle Briana , Giles Jennifer , VerMeer Mark 
Published: November 2023

The G protein‐coupled estrogen receptor (GPER/GPR30) participates in various Ca2+‐dependent cellular functions, yet its direct effects and mechanisms on components of Ca2+ signaling machinery are incompletely understood. Store‐operated Ca2+ entry (SOCE) is an essential Ca2+ signaling mechanism that regulates numerous cellular activities. Here we show that GPER/GPR30 expression and activation limits SOCE. Heterologous expression and gene silencing studies demonstrated that GPER/GPR30's expression puts a constitutive break on SOCE. Co‐immunoprecipitation and confocal microscopy studies indicated that GPER/GPR30 interacts with the stromal interaction molecule 1 (STIM1) at different interaction sites in distinct subcellular compartments. Pharmacological studies indicated that GPER/GPR30 activation acutely further suppresses SOCE in addition to the effect of physical interaction between GPER/GPR30 and STIM1. Site‐directed mutagenesis studies indicated that GPER/GPR30 activation inhibits SOCE via alteration in the phosphorylation status of STIM1. Taken together, the data demonstrate physical and functional interactions as mechanisms whereby GPER/GPR30 expression and activation regulates SOCE.Support or Funding InformationNIH HL112184 (to KT)
A G Protein‐Biased Ligand of the Kappa Opioid Receptor is Antinociceptive and Antipruritic But Does Not Cause Sedation or Dysphoria
Authors: Brust Tarsis F , Morgenweck Jenny , Kim Susy A , Rose Jamie H , Locke Jason L , Schmid Cullen L , Zhou Lei , Stahl Edward L , Cameron Michael D , Scarry Sarah M , Aubé Jeffrey , Jones Sara R , Martin Thomas J , Bohn Laura M 
Published: November 2023

The kappa opioid receptor (KOR) has been pursued as a target to treat pain and pruritis (itch). In contrast to opioid narcotics that target primarily the mu opioid receptor, KOR agonists are devoid of addictive and life‐threatening side effects, such as respiratory depression. However, the clinical use of KOR agonists has been limited by the onset of dysphoria and sedation. The KOR is a G protein‐coupled receptor (GPCR) that mediates its physiological actions through the activation of multiple signaling effectors, such as G proteins and βarrestins. Notably, the molecular pharmacology of GPCRs can be specifically modulated by functionally selective (or biased) ligands to induce preferential activation of a subset of the receptor's responses. The use of such ligands has been proposed as a strategy to improve drug therapies targeting GPCRs. For the KOR, selective activation of G proteins over βarrestins may present a means to promote analgesia and inhibit itch in the absence of dysphoria and sedation. In the current study, we evaluate a recently developed G protein‐biased KOR agonist in preclinical models of pain, pruritis, sedation, dopamine regulation and dysphoria. We show that while triazole 1.1 is antinociceptive and antipruritic, the compound does not induce sedation in mice. Additional studies show that, in contrast to the reference KOR ligand U50,488H, triazole 1.1 does not cause reductions in dopamine release or behaviors indicative of dysphoria in rodent models. These results illustrate that biased ligands may be useful to separate physiological responses downstream of GPCRs. Furthermore, our findings suggest that G protein‐biased ligands at the KOR, which inherently have reduced abuse potential, may present a means to treat pain and intractable itch without the side effects of dysphoria and sedation.Support or Funding InformationThis work was supported by NIH grants P01GM113852 and P50DA006634 (to T.J.M.), R01DA014030 and U01AA014091 (to S.L.J.), and R01DA031297 (to L.M.B. and J.A.).
Mechanistic Classification of Toxicants Using Differential Impedance‐Based Real Time Cytotoxicity Profiles in Primary Human Hepatocytes
Authors: Liguori Michael J , Sharapova Tatiana , LeRoy Bruce , Blomme Eric AG 
Published: November 2023

Impedance‐based real time cytolethality profiling has become an important lead optimization toxicology tool in pharmaceutical discovery. This technique offers the advantage of continuous, non‐destructive monitoring of changes in electrical impedance (reported as cellular index or CI) that correlates to alterations in cell health, morphology, and proliferation of adherent primary cells as well as cell lines. In this study, we used a panel of reference toxicants representing several distinct mechanisms of hepatotoxicity and evaluated their impact on CI profiles of cultured primary human hepatocytes (PHH). The mechanisms of action included: apoptosis (staurosporine; ceramide c2), cytoskeletal disruption (phalloidin; colchicine), mitochondrial inhibition (antimycin A; carbonylcyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP)), plasma membrane perforation (Triton X‐100), and general cellular detachment (trypsin digestion). PHH donor to donor variability (n= 6 donors) was assessed using chlorpromazine, which acts through two cytotoxicity mechanisms: oxidative stress via quinoneimine formation and changes in mitochondrial membrane potential. Cryopreserved PHH were thawed and cultivated using conventional conditions in Williams E‐based media. Cells were treated with eight concentrations of each toxicant or vehicle (0.1 to 0.5% DMSO) in electrode coated 96‐well plates and monitored with an xCELLigence real‐time cell analyzer (Acea Biosciences) for up to 3 days. Treatment of PHH with surfactant (Triton X‐100) or trypsin resulted in an immediate decline of normalized cellular index (NCI). Mitochondrial function inhibitors caused a rapid decrease of NCI at doses as low as 1.6 and 3.1 μM. Staurosporine and ceramide treatment (apoptosis inducers) decreased NCI (IC50 =1 to 6 μM), but over an extended time (>10 h) compared to the mitochondrial toxicants. Interestingly, exposure to cytoskeletal disruptors led to a gradual increase in NCI over 3 days, plausibly due to subtle alterations in cell shape. There was a high degree of inter‐individual variability in response to chlorpromazine treatment suggesting that cryopreserved PHH should be characterized using a positive control before an impedance‐based compound evaluation. Altogether, these results are useful to understand differences in impedance profiles based on compound toxicity mechanisms and can be compared to profiles from related liver‐derived primary cells and cell lines.Support or Funding InformationAll authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.
Blood Pressure in the SHR Following Renal Denervation or Dopamine β‐Hydroxylase Inhibition with Etamicastat
Authors: Pires Nuno , Igreja Bruno , Moura Eduardo , Wright Lyndon , Serrão Paula , Soares‐da‐Silva Patrício 
Published: November 2023

Overactivity of the sympathetic nervous system plays an important role in the development and progression of arterial hypertension. Catheter‐based renal nerve ablation for treatment of drug‐resistant hypertension has been recently developed. An alternative strategy for modulation of sympathetic nerve function is to reduce the biosynthesis of norepinephrine (NE) via inhibition of dopamine β‐hydroxylase (DβH), the enzyme that catalyses the conversion of dopamine to NE in sympathetic nerves.Renal denervation (RDN) was performed in spontaneously hypertensive rats (SHR) to evaluate its effect on NE levels and blood pressure over a 28‐day period. Chronic oral treatment with the peripherally selective DβH inhibitor etamicastat (30 mg/kg/day) was performed in another cohort of SHR.RDN and chronic oral treatment with etamicastat did not affect renal function, as assessed by urinary protein, creatinine and urea levels. RDN significantly decreased mean arterial blood pressure (MAP, in mm Hg) 7 days post‐surgery (139±6, P<0.01) versus baseline values (158±6). A gradual return of MAP to high baseline levels was observed over time; 139±7 at day 14, 151±12, at day 21 and 155±12 at day 28 after RDN. Treatment with etamicastat decreased baseline MAP (150±5) in a sustained and significant (P<0.01) manner at all‐time points: 141±5 at day 7, 139±5 at day 14, 138±5 at day 21 and 143±5 at day 28. At day 28, NE levels (in pmol/mg tissue) in renal tissue were significantly decreased in both RDN (0.12±0.08, P=0.001) and etamicastat‐treated groups (0.48±0.04, P=0.01) as compared to vehicle group (0.88±0.13). In contrast, left ventricle NE levels were decreased after treatment with etamicastat (1.24±0.12, P=0.04) but not in the RDN group (2.53±0.24, P=0.9) when compared to controls (2.59±0.58).It is concluded that RDN produces transitory decreases in MAP, whereas prolonged down regulation of sympathetic drive with the DβH inhibitor etamicastat results in sustained decreases in MAP.Support or Funding InformationThis work was supported by BIAL ‐ Portela & Ca, S.A.
Improving Therapy for Refractory Mantle Cell Lymphoma Using Small Molecule Inhibitors Vorinostat and Palbociclib to Target Histone Deacetylase and Cyclin Dependent Kinase 4/6
Authors: Hatch Nathan , Chaturvedi Nagendra , Kling Mathew , Joshi Shantaram 
Published: November 2023

Mantle Cell Lymphoma is a B‐Cell non‐Hodgkin lymphoma that accounts for 10% of all non‐Hodgkin lymphoma cases. It is extremely aggressive with a median survival time of approximately 5 years. There is a high rate of relapse due to therapy‐resistant tumor cells. There is currently no cure for refractory MCL. Here we demonstrate that using the histone deacetylase (HDAC) inhibitor, Vorinostat, in conjunction with cyclin dependent kinase (CDK) 4/6 inhibitor, Palbociclib, can be an effective method of treatment. This is shown using multiple in vitro experiments including: MTT assay, Annexin‐V staining, and colony formation assay. This combination of small molecule inhibitors has been found to decrease tumor cell proliferation by at least 25% as well as increase the rate of tumor cell apoptosis by at least 60% when compared to the use of these drugs on their own at equivalent concentrations. Future research will consist of in vivo studies using murine models.Support or Funding InformationThis publication was made possible by grants from the Lymphoma Research Foundation New York, NY, the State of Nebraska LB506 Funds, and the National Institute for General Medical Science (NIGMS) (8P20GM103427), a component of the National Institutes of Health (NIH) and its contents are the sole responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.
Employing Surface‐Displayed Proteins on Bacillus megabacterium Spores to Detect Environmental Pollutants
Authors: Wynn Daniel Toner , Mittal Rhiya , Knecht Leslie , Deo Sapna , Daunert Sylvia 
Published: November 2023

Arsenic poses a major threat to both human populations and the environment. It is carcinogenic and a potent toxin. Because of its hydrophilicity, dangerous levels of arsenic can be found in the drinking water of many communities, especially in areas where water is collected from wells. As such, developing methods to quickly and reliably assess a wide variety of matrices for the presence of arsenic is very important. The current gold standard for arsenic detection is inductively‐coupled plasma‐mass spectrometry (ICP‐MS). Due to the limitations of ICP‐MS, chiefly its expense and inconvenience, many other methods have been developed, including biosensors.We have developed a novel biosensor that employs the morphology of bacterial endospores to create spores engineered to be analytical tools (SEATs). In these biosensors, the sensing and reporting elements are displayed on the surface of Bacillus megaterium spores. This was accomplished by creating a triple fusion protein that joins the spore coat protein CotB, the arsenite binding protein ArsR, and the fluorescent reporter EGFP N‐terminus to C‐terminus, respectively. SEATs have greater sensitivity than traditional cell based biosensors, with limits of detection of 1 × 10‐11 M. We have also demonstrated that SEATs are suitable for use in complex matrices using both spiked samples and samples collected from local areas with arsenic contamination.Support or Funding InformationFunded by the National Science Foundation.
Determination of total oxytocin in human serum by traditional LCMS
Authors: Franke Adrian , Menden Ariane , Li Xingnan 
Published: November 2023

Oxytocin (OT) is a nanopeptide with hormone, neurotransmitter/neuromodulator and other activities. Interest in measuring OT has peaked in recent years due to its newly reported association with autism, schizophrenia, depression, social affiliation and many other conditions. Non‐protein bound (‘free’) OT is present in the human circulation but its measurement using traditional ELISA has been unreliable and inconsistent among different manufacturers partly due to its low innate concentrations (low pg/mL).Liquid chromatography mass spectrometry (LCMS) represents an attractive alternative to ELISA, however, accurate measurements in the desirable concentration range often require labor intensive sample preparation and ultra‐sensitive expensive LCMS technology–issues which can be bypassed by simultaneously analyzing bound and unbound (total) OT levels, which are typically in the ng/mL range. Braendtzag et al. (2016, Nature: Scientific reports) measured total OT after chemical reduction and alkylation (R/A) prior to on‐line C18 solid‐phase extraction (SPE) and nano LCMS. This specialized setup was simplified in our method by applying a weak cation‐exchange column (Strata‐X‐CW 33 μm; 30 mg/ 1 mL) Phenomenex) for simple off‐line SPE purification of the R/A product followed by LCMS (orbitrap model Q‐Exactive, Thermo Scientific) using a Kinetex pre and analytical C18 column (5 × 3.0 mm, 2.6 μm and 150 × 3.0 mm, 2.6 μm; Phenomenex) and electrospray ionization followed by targeted selected ion monitoring MS in positive mode. Validation of our method using pooled serum samples (premenop. women) resulted in an intra‐ and inter‐day CV of 2.6 – 3.2 % and 2.9 % (1.67 ng/mL), and 0.7 – 11.9 % and 8.3%, respectively, for this pool spiked with 1 ng/mL with an average accuracy of 104% (n=3). The LLOD (S/N=3) and LLOQ (S/N=10) using 200 μL serum was 0.01 ng/mL and 0.033 ng/mL, respectively. Total OT levels in serum from healthy non‐pregnant young females (n=3), one healthy male as well as human pooled commercial serum from both genders (n=2) or a pool of female samples (n=2), ranged from 0.23–8.4 ng/mL with a median level of 1.78 ng/mL.Our method for total OT in serum represents an attractive alternative to traditional ELISAs and labor‐intensive LCMS techniques as it is accurate, precise, simple, and requires MS instrumentation typically found in an analytical chemistry laboratory. It requires only 200 μL serum and can be easily applied to investigate OT as a biomarker, treatment agent and target in psychological disease plus provides an inexpensive tool to investigate OT's connections to other functions.Support or Funding InformationP30 CA71789
Dissecting the Dual Activity of Cellular Retinoic Acid Binding Protein 2 (CRABP2)
Authors: Vreeland Amanda , Driscoll Donna , Noy Noa 
Published: November 2023

Cellular retinoic acid‐binding protein 2 (CRABP2) displays two distinct functions. The classical function of this protein is to directly deliver retinoic acid (RA) to retinoic acid receptors (RARs), thereby enhancing the transcriptional activation of RARs and upregulating expression of target genes, including some involved in suppression of tumor growth. We have recently elucidated a second function of CRABP2, which is exerted in the absence of RA. We found that apo‐CRABP2 cooperates with the RNA‐binding and stabilizing protein HuR to upregulate and stabilize mRNAs. CRABP2 directly interacts with HuR and enhances its affinity for target mRNAs, including some involved in regulating oncogenic properties. Notably, while apo‐CRABP2 is cytosolic and binds to HuR, the complex dissociates upon binding of RA. Holo‐CRABP2 then translocates to the nucleus where it delivers RA to RAR. CRABP2 then returns to the cytosol and re‐assocaites with HuR, showing that CRABP2 is able to perform both of its functions in parallel. The goal of this work is to determine the structural charcteristics of CRABP2 that mediate it's interaction with HuR.The RA‐responsiveness of the interaction between CRABP2 and HuR suggests that the residues on CRABP2 that stabilize these interactions can “sense” ligand‐binding, i.e. that they undergo conformational changes upon ligation of CRABP2. Analysis of the reported three dimensional X‐ray crystal structures of holo‐ and apo‐CRABP2 revealed two surface residues, Arg60 and Lys107, whose side chains are placed at different conformations in the absence and presence of the ligand. We have found that Arg60 is necessary for the RA‐responsiveness of the CRABP2‐HuR complex and mutation of this residue prevents CRABP2 from cooperating with RAR. Additionally, we have found that Lys107 is necessary for CRABP2 to cooperate with HuR, but mutation of this residue does not interfere with the ability of CRABP2 to cooperate with RAR. As CRABP2 displays tumor suppressive activities, current work is examining which of CRABP2's functions is responsible for the tumor supressive activity of the protein.Support or Funding InformationThis work was supported by NIH grant R01‐CA166955. A.V. was partially supported by NIH grant R25‐CA148052.