bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evaluation of Immunohistochemical Markers for Application in a Novel Neurofibromatosis‐1 Porcine Model
Authors: Meyerholz David K , Ofori‐Amanfo Georgina K , Leidinger Mariah R , Quelle Dawn , Darbro Benjamin , Panzer Karin , Sieren Jessica C , Khanna Rajesh , Rogers Chris , White Katherine , Weimer Jill 
Published: November 2023

Neurofibromatosis type 1 (NF1) is a genetic condition caused by mutations in the tumor suppressor gene, NF1. People with NF1 have an increased risk for benign and malignant tumors of the CNS/PNS (e.g. neurofibromas, malignant peripheral nerve sheath tumors, gliomas) and other tumor types (e.g. leukemias, breast cancer, etc.). Additionally, those affected by NF1 are susceptible to other clinical manifestations including (but not limited to) learning disabilities, pain/migraines, hypertension, café au lait macules, and developmental lesions (e.g. tibial pseudoarthrosis). Translational study of NF1 has been constrained, in part, by the failure of animal models to faithfully recapitulate the scope of phenotypes seen in patients. Recently, we have developed a porcine model of NF1 and early phenotypic analysis suggests it will be useful for study. Nonetheless, one potential hurdle to porcine models of human disease has been the lack of validated tools and reagents for translational study. We used formalin‐fixed human and wild type porcine tissues to evaluate select immunohistochemical markers of CNS/PNS tissues (calbindin D, glucose transporter 1, ionized calcium binding adaptor molecule 1, glial fibrillary acidic protein, laminin, myelin basic protein, beta‐tubulin III, Von Willebrand factor and S100), immune cells (CD13, CD20) and cell cycle regulation (activated caspase‐3, proliferating cell nuclear antigen). We optimized and validated each technique in human and porcine tissues using cells/tissues with known marker expression for anatomical reference. Remarkably, the optimized techniques were frequently identical for human and porcine tissues, and when not the case, the technique often required only slight modification of the primary antibody concentration. These findings demonstrate a commonality in marker expression and distribution between species and also show the utility of these markers for future translational studies in the novel NF1 pig model.Support or Funding InformationChildren's Tumor Foundation
Visualization of neuronal connectivity in NPARM PHOX2B mutants with a modified PACT protocol
Authors: Fair Summer Rose 
Published: November 2023

NPARM PHOX2B mutations represent a subset of congenital central hypoventilation syndrome (CCHS). To understand the abnormal innervation of breathing column nuclei in CCHS, it is necessary to visualize hindbrain axonal projections and stereologically quantify differences in their connectivity. The task of visualizing neuronal connectivity typically relies on stereotactic injection of retrograde tracers; this method is often tedious, subject to inaccuracy and yields a low number of experimental subjects. When prepared for analyzation, the lipid molecules in the tissue inhibit the diffusion of immunofluorescent antibodies into tissue sections, thereby preventing entire visualization of target proteins in tissue. The present study proposes a twofold solution for the current methodology of neuronal connectivity visualization with the removal of lipid molecules from several transgenic, developmental tracer models to identify anatomical differences in the breathing column. Lipid molecules are removed from the tissue sections in a two phase process adapted from a Passive Clarity Technique (PACT) protocol. First, sections are cross‐linked and hybridized to hydrogel monomers. Next, the lipids are washed from the tissue bound to the monomers with a detergent [8% SDS]. The tissue sections are then stained with immunofluorescent antibodies, imaged using confocal microscopy and analyzed with stereology to quantify differences in nuclei innervation. This approach yields high quality images of the cell bodies and axons of Phox2b derived cells. This novel method of visualizing neuronal connectivity can be implemented in a diverse spread of studies to improve identification and morphology of neurons in several disease states.Support or Funding InformationFunding provided by the R01 MECHANISMS OF CONGENITAL HYPOVENTILATION (Project # 3R01HL132355‐01A1S2)
Hands‐on Modeling/Simulation Using MATLAB and SimBiology Improves Student Learning in Graduate Pharmacology and Pharmaceutical Sciences Education
Authors: Chang JuOae , Alsulimani Helal Hussain , Han Murui , Ye Qi , Buyukozturk Fulden , Kim Jonghan 
Published: November 2023

Quantitative characterization of pharmacokinetics (PK) and pharmacodynamics (PD) of drugs is an essential component in pharmacology/toxicology education as well as drug discovery/development. PK determines the relationship between the dose of the administered drug and the concentration measured in the body, whereas PD characterizes the extent of pharmacological effect induced by drug concentrations. Simulation and modeling of such properties is a powerful tool to predict drug's efficacy and toxicity with the limited dose‐concentration‐response data. Several software tools, such as WinNonlin, Simcyp, GastroPlus and SAAM II, are traditionally used for PK/PD modeling and simulation. However, these tools have not demonstrated the dynamic flexibility in the generation of novel modeling approaches in both academic and research settings. While MATLAB has been extensively used in the field of Science and Engineering with a variety of applications, SimBiology provides an app to model, simulate, and analyze dynamic systems, focusing on PK/PD and quantitative systems pharmacology (QSP) applications. It also provides a block diagram editor for building models, or the user can create models programmatically using the MATLAB language. Moreover, SimBiology includes a library of common PK models, which one can customize and integrate with mechanistic systems biology models. Consequently, MATLAB/SimBiology provides a comprehensive tool that can help to train young scientists in the field of pharmaceutical sciences, including pharmacology and toxicology. While our graduate program at Northeastern University offers several important features of fundamental PK/PD principles and advanced PK/PD analyses, an opportunity to have a hands‐on experience on PK/PD simulation and modeling has not been provided. In Spring 2016, we incorporated SimBiology as a modeling and simulation tool to our graduate Advanced Pharmacokinetics and Toxicology course through a collaboration with MathWorks. We then assessed the benefits of SimBiology in students' learning based on class participation and problem sets/exams, as well as students' survey through Teacher Rating and Course Evaluation (TRACE). The results demonstrated that SimBiology improved students' learning in several TRACE sections, including “The classroom technology helped me to learn”, “I found this course intellectually challenging”, and “I learned a lot in this course”. Our results also indicate the benefits and importance of hands‐on experience in modeling software to better understand PK/PD in pharmacology.Support or Funding InformationThis project was supported by MathWorks Curriculum Development Grant.
Effects of sequential exposure to ethanol and methamphetamine on tissue contents of dopamine, serotonin and glutamate in Wistar rats
Authors: Almalki Atiah H. , Das Sujan C. , Alshehri Fahad S. , Althobaiti Yusuf S. , Sari Youssef 
Published: November 2023

Compulsive ethanol intake and methamphetamine (METH) are considered as a major public health issue. Ethanol and METH exposures have been associated with changes in neurotransmitters levels in several central brain regions. However, there is little known about the co‐abuse of ethanol and METH on glutamate, GABA, dopamine and serotonin tissue contents in striatum and hippocampus. In this study, we investigated the effects of sequential exposure to ethanol and METH on these neurotransmitters. Young adult male Wistar rats were orally gavaged with either ethanol (6 g/kg) or water for seven days. On Day 8, rats were administered high dose of METH (10 mg/kg, i.p. every 2 h× 4). We revealed additive effects of ethanol and METH on the reduction of dopamine tissue contents in striatum as well as reduction of serotonin tissue contents in striatum and hippocampus as compared to each drug administered alone. Moreover, glutamate concentrations were increased in ethanol and METH group compared to ethanol administrated alone but decreased compared to METH administered alone group in striatum. These findings suggest that ethanol has an additive effect to METH on several neurotransmitters tissue contents in striatum and hippocampus.Support or Funding InformationThis work was supported in part by Award Number R01AA019458 (Y.S.) from the National Institutes on Alcohol Abuse and Alcoholism and by fund from the University of Toledo.
PARP mediates binge alcohol‐induced neurodegeneration in adult rats
Authors: Kouzoukas Dimitrios E. , Schreiber Jennifer A. , Tajuddin Nuzhath F. , Kaja Simon , Kim Hee‐Yong , Neafsey Edward J. , Collins Michael A. 
Published: November 2023

Chronic alcohol (ethanol) abuse damages brain structures associated with learning and memory. This clinical neuropathological finding is corroborated by our observations of selective hippocampal (HP) and entorhinal cortical (EC) neurodegeneration in vivo in a model of repeated binge alcohol intoxication in adult rats (Majchrowicz 1975), and in vitro in repetitive binge alcohol‐treated rat adult‐age HP‐EC slice cultures (Tajuddin et al. 2014). We previously reported that 1) neurodegeneration in these two models correlated with elevated levels of a DNA repair protein, poly [ADP‐ribose] polymerase‐1 (PARP‐1), and 2) pharmacological PARP inhibition blocked the neuronal damage in the slice cultures. Despite PARP‐1's DNA repair role, its overactivation is also known to trigger parthanatos, a form of regulated neuronal necrosis. To examine the relationship in vivo between PARP activation and alcohol‐induced neuronal injury, we tested whether blocking PARP activity affects HP and EC neurodegeneration, quantified by Fluoro‐Jade B staining, in binge alcohol‐intoxicated adult male Sprague Dawley rats (above‐mentioned Majchrowicz model). Rats received alcohol (0 to 5g/kg i.g., determined by intoxication score) or dietary control every 8 hours for 4 consecutive days, with or without the PARP inhibitor, veliparib (ABT‐888; 25 mg/kg/day i.g.). Both alcohol‐treated groups had similar peak blood alcohol levels of ~360 mg/dl (~80 mM). PARP inhibition significantly suppressed the alcohol‐induced neurodegeneration in the HP dentate gyrus and the lateral EC region by 79.1% and 66.3%, respectively. These results confirm that PARP activation plays a crucial role in neurodegeneration (presumably regulated necrosis) caused by repeated severe binge alcohol intoxication. Furthermore, they provide key support for the validity of our in vitro adult‐age slice culture model of binge alcohol‐induced neuronal damage.Support or Funding InformationNIH U01 AA018279 (MAC) and NIH T32 AA013527 (LUMC Alcohol Research Program)
Interactive Effects of Hormone Status and Δ9‐THC Administration on Memory in Female Rats
Authors: Fournett Alyssa Christine , Winsauer Peter 
Published: November 2023

The cannabinoid receptor agonist, Δ9‐tetrahydrocannabinol (Δ9‐THC), is a psychoactive constituent of marijuana, and a popular recreational drug that has been shown to produce disruptions in learning and memory. Conversely, estrogen has been found to produce a positive effect on learning and memory in female rats. The purpose of this study was to examine the effects of hormone status on memory, and how these effects alter the acute disruptive effects of Δ9‐THC (0.32–3.2 mg/kg) on memory in female rats. To do this, intact and ovariectomized (OVX) female rats were trained on a repeated acquisition and delayed‐performance procedure. During the acquisition phase of this procedure, rats acquired a 4‐response sequence (CRLC, LRCL, RLCR, etc.) that changed daily. Responding was maintained under a second‐order fixed‐ratio 3 schedule of food presentation. Sequence acquisition was followed by a delay phase and a delayed‐performance phase in which rats emitted the previously acquired sequence. Delay phase under baseline conditions was 1 hour. In order to examine the effect of delay, delays ranging from 1 minute to 24 hours were used. Δ9‐THC or vehicle was administered 30 minutes prior to the delayed‐performance phase in order to specifically target retention, which was assessed by percent savings. Response rate and the percentage of errors were also recorded. When the delay was varied under non‐drug conditions, delay‐dependent decreases in percent savings were observed in intact and OVX rats. However, the disruption of retention was greater in OVX than intact rats. Acute administration of Δ9‐THC produced dose‐dependent decreases in response rate and percent savings, and increases in percent errors in both intact and OVX rats under baseline conditions. After administration of 0.56 mg/kg Δ9‐THC, intact rats showed less sequence retention than OVX rats. Increasing the delay to 3 hours produced a leftward shift in the percent‐savings curve for both intact and OVX rats. These results suggest that hormone status directly impacts retention under an acquisition and delayed‐performance procedure, and acute administration of Δ9‐THC produces both delay‐dependent and dose‐dependent effects on memory in intact and OVX female rats.Support or Funding InformationNIH grant R01‐DA037255‐02S1
Combined Norepinephrine and Dopamine Reuptake Inhibition for Long‐Term Memory Enhancement
Authors: Pantoni Madeline M , Anagnostaras Stephan G 
Published: November 2023

The psychostimulants amphetamine and methylphenidate are highly effective cognitive enhancers, particularly for their ability to enhance long‐term memory (LTM). While psychostimulants have been argued to exert their procognitive effects exclusively through norepinephrine transporter (NET) inhibition and their reinforcing effects exclusively through dopamine transporter (DAT) inhibition, there is increasing evidence that DAT also plays a critical role in learning and memory, including LTM enhancement. In the present study, we examined the effects of a drug combination of a potent NET inhibitor with a weak DAT inhibitor, using Pavlovian fear conditioning. While NET or DAT inhibition alone failed to enhance LTM across a wide range of doses (Carmack et al., 2014), we found that combined NET/DAT inhibition produced robust enhancements in immediate and long‐term fear memory acquisition. These findings support the theory that both NET and DAT inhibition are required for psychostimulant‐induced LTM enhancement. Additionally, we propose that a pharmaceutical combination of moderate NET inhibition and weak DAT inhibition could lead to the development of a non‐stimulating, non‐addictive, and non‐controlled, yet highly effective cognitive enhancer.Support or Funding InformationNIH grant DA020041 (SGA)
Elucidating Functional Selectivity at the 5‐HT2A Receptor
Authors: Billac Gerald , Nichols Charles 
Published: November 2023

There has been a recent resurgence of clinical interest in the application of psychedelic drugs for the treatment of various disease states. While the field is mostly focused on action within the central nervous system, previously published data from our lab supports the use of these 5‐hydroxytryptamine 2A receptor (5‐HT2AR) agonists as modulators of the inflammatory pathway in the periphery – with potential applications ranging from treatment to prevention of a spectrum of inflammation‐based diseases (e.g., asthma, atherosclerosis, Alzheimer's, Crohn's, etc.). The ligand selected for further analysis in our in vivo models of asthma and atherosclerosis, (R)‐2,5‐dimethoxy‐4‐iodoamphetamine [(R)‐DOI], demonstrates superpotent anti‐inflammatory characteristics. Whereas these whole organism models will provide insight into the physiological response profile, the mechanism of action and structural basis of the anti‐inflammatory effects have yet to be elucidated. Examining a panel of 5‐HT2AR ligands, we are elucidating the differential activation of effector pathways with particular focus on the structure‐activity relationship (SAR). Initial results from examination of the canonical G〈q pathway using a calcium mobilization measure show an experimentally validating response for the endogenous ligand serotonin, as well as, exhibit ligand‐dependent differential activation. Interestingly, (R)‐2C‐BCB showed a significant increase in percent calcium mobilization versus the remainder of the panel, which suggests the possibility that it is strongly biased towards the G〈q pathway. Ongoing studies are examining ®‐Arrestin signaling via a comparative analysis of ERK phosphorylation, ®‐Arrestin recruitment through a luciferase reporter gene assay, receptor internalization kinetics utilizing Fluorogen Activating Peptide (FAP) technology in a fluorescence depletion assay, and any additional GPCR signaling (Gi/o/s) via a comparative analysis of cAMP accumulation. The collective analysis of a comprehensive panel of 5HT2AR ligands has never been examined in this fashion; thus, we hope to apply the generated data and SAR to develop novel molecular therapies targeting specific effector pathways.Support or Funding InformationEleusis PBC
Leveraging a Novel ITIM Motif in GPCRs for Targeted Antibody Design
Authors: Belcastro Lili T. , Jancina Anastasia , Adams Christina , Paulukinas Ryan D. , Moore Catherine C. 
Published: November 2023

CXCR4, a chemokine GPCR, is essential for migration of neuronal, hematopoietic, and breast cancer cells during metastasis whereby CXCR4 dysregulation promotes migration and invasion. Following SDF stimulation, CXCR4 is phosphorylated on Ser/Thr residues which initiates adaptor recruitment, receptor desensitization, and trafficking to endocytic sites. Here we show that stimulation with gradient SDF, delays receptor phosphorylation and trafficking, leading to sustained signaling to a novel CXCR4‐SHP2‐ERK pathway. SHP2 is a tyrosine phosphatase implicated in HER2(+) and triple‐negative breast cancers, whereby it transduces mitogenic and migratory signals driving hyperproliferation and invasion. SHP2 is recruited to tyrosine phosphorylated ITIM motifs (immmunoreceptor tyrosine‐based inhibitory consensus motifs), a hallmark found in inhibitory immune receptors with little evidence in GPCRs. Here we identify an ITIM motif in CXCR4 that regulates both SHP2 binding and signaling. Specifically, we assessed if gradient SDF stimulation of CXCR4 1) delays receptor phosphorylation and trafficking, 2) sustains signaling to SHP2‐ERK, 3) induces SHP2‐dependent migration; and if CXCR4 Tyr mutation within the ITIM motif 4) maintains SDF gradient sensing ability, and 5) disrupts interaction with and signaling to SHP2. Our data demonstrate that gradient SDF delays receptor Ser/Thr phosphorylation and internalization thereby sustaining signaling to SHP2‐ERK and driving SHP2‐dependent migration. Furthermore, the ITIM mutant maintains SDF gradient sensing ability, but disrupts interaction with and signaling to SHP2. Our data support a working model that CXCR4 contains a functional ITIM motif which we are currently leveraging for targeted antibody design for use in migration studies of aggressive breast cancer cells with dysregulated CXCR4.Support or Funding InformationThese studies were supported by NIH grant GM‐097718, PA Department of Health grant SAP4100057688, and the Milton Lev Memorial Faculty Research Fund.
Optimization of a Novel D2 Dopamine Receptor Antagonist Scaffold Reveals Exceptionally Selective and Potent Lead Molecules
Authors: Day Marilyn M. , Dulcey Andres E. , Bayron Diane , Stang Christopher R.T. , Moritz Amy E. , Doyle Trevor , Javitch Jonathan A. , Southall Noel T. , Ferrer Marc , Marugan Juan J. , Sibley David R. , Free R. Benjamin 
Published: November 2023

FDA‐approved antipsychotics are primarily D2 dopamine receptor (D2R) antagonists, however they generally lack receptor selectivity and interact with other GPCRs, including D3Rs. Despite its clear clinical importance, there are few compounds that selectively modulate the D2R. This is due to structural similarities in the orthosteric binding sites of the D2‐like receptors (D2R, D3R, and D4R), as well as other biogenic amine receptors. To find better therapeutic candidates, as well as probe compounds to investigate D2R pathophysiology, we sought to identify highly selective D2R antagonists by implementing a high‐throughput screening (HTS) campaign using more than 400,000 unique compounds in the Molecular Libraries Screening Center Network library. Most of the hit compounds that were identified as antagonists of the D2R were also antagonists of the closely related D3R. However, several compounds were found to exhibit high affinity for the D2R with low affinity for the D3R. One such compound (MLS6916) served as our lead scaffold for optimization. MLS6916 is ~700 fold selective for the D2R vs. D3R as determined in functional β‐arrestin recruitment assays and radioligand binding competition assays. Importantly, MLS6916 was also examined for functional activity at 168 different GPCRs and showed no agonist activity on any GPCR tested, and only exhibited antagonist activity at the D2‐like receptors. Preliminary investigations, however, revealed that this initial hit compound was metabolically unstable. To optimize this hit scaffold, we conducted an iterative medicinal chemistry campaign with the goals of increasing selectivity, potency, and engendering metabolic stability. Over 70 analogs of the MLS6916 parent compound were synthesized to dissect the influence of various constituents of the molecule and to construct structure‐activity relationships. Analogs were analyzed for activity on D2R, D3R, and D4R using both radioligand binding and functional assays to determine affinities and potencies for the D2‐like receptors. In addition, analogs were tested for solubility, permeability, and stability. Several lead compounds were identified with Ki values of < 50 nM for the D2R with > 1,000‐fold selectivity vs. the D3R. These selectivity trends were confirmed in functional β‐arrestin recruitment assays. Importantly, lead compounds with enhanced metabolic stability and permeability were identified. Further analysis and optimization of these lead compounds will provide new insight into highly selective D2R antagonists for the treatment and understanding of dopamine‐related disorders.Support or Funding InformationNINDS Intramural Research Program