bluuebunny/crossref_metadata_2025 huggingface dataset:
Phospholipase D Regulates GSK3β Mediated Epithelial to Mesenchymal Transition and Akt Mediated Cell Death Leading to Pulmonary Fibrosis
Authors: Suryadevara Vidyani , Royston Thomas J. , Natarajan Viswanathan
Published: November 2023

Idiopathic pulmonary fibrosis (IPF) is a pernicious lung disease characterized by scar formation and respiratory failure. This is due to dysregulated repair of the epithelial injury in the lung, which involves epithelial apoptosis and also epithelial to mesenchymal transition (EMT), thereby leading to the deposition of the extracellular matrix in the lung. Our previous studies have established a role for phospholipase D (PLD) as genetic deletion of PLD2 in mice conferred protection against bleomycin induced PF. PLD2−/− mice exhibited less inflammation on day 7 and less pulmonary fibrosis on day 21 post‐bleomycin challenge compared to wild type mice. This study aims at understanding how PLD regulates GSK3β mediated EMT and Akt mediated apoptosis. GSK3β and Akt are known to be involved in crucial signaling pathways that regulate cell death and survival. To elucidate the role of PLD signaling in epithelial injury, Beas2B cells were pre‐incubated with either PLD1 (VU0155069‐ 250nM), PLD2 (VU0364739‐500nM) or PLD1 +PLD2 inhibitors prior to bleomycin challenge. Bleomycin challenge enhanced phosphorylation of GSK3β at Ser9 leading to its inactivation, which was reversed by inhibition of PLD1 and PLD2. Also, activated Akt as shown by enhanced phosphorylation at Ser473 was reversed upon PLD1 and PLD2 inhibition. Inhibition of PLD1 and PLD2 was necessary to attenuate bleomycin mediated apoptosis of Beas2B cells as evidenced by cleavage of Caspase‐3 and PARP. The results presented here suggest that PLD regulates bleomycin‐induced pulmonary fibrosis by modulating apoptosis and EMT via Akt/GSK3β signaling in the epithelial cells, and targeting PLD may be beneficial.

Support or Funding Information

This work is supported by National Institutes of Health grant P01 HL 98050 to VN.
Detection of Phenotypic Differences in Alveolar Macrophages Using Ionized Calcium‐Binding Adapter Molecule 1 Marker
Authors: Meyerholz David K , Hogan Christin M. , Glanz Ryan M , Goeken J Adam , Leidinger Mariah R , Ofori‐Amanfo Georgina K , McQuillen Logan , Donovan Kathleen M , Harwani Sailesh C , Flaherty Heather A.
Published: November 2023

Alveolar macrophages are resident immune cells in the distal lung and offer a first line of defense to inhaled pathogens and environmental particulates. In healthy states, these resident immune cells reside in the alveolar spaces of the lung as quiescent monitors, but upon stimulation, they play significant roles in host defense, removal of inhaled particulates and tissue remodeling. Upon activation, alveolar macrophages can exhibit morphological changes including increases in cell size, ruffled borders and altered expression of markers. In this study of 10 week old male rats, we had immunostained lung sections for the marker, ionized calcium‐binding adapter molecule 1 (IBA1). We evaluated whether quantitative or qualitative differences could be detected in alveolar macrophages between the normotensive WKY (Wistar Kyoto) strain and the hypertensive SHR (Spontaneous Hypertensive Strain) strains. For all analysis, statistical significance was set at P<0.05. Immunocytochemistry of bronchoalveolar fluid showed similar immunostaining of macrophages in both groups with no overt group‐distinguishing features. However, examination of immunostained paraffin embedded tissue sections suggest that SHR lungs have subpopulations of macrophages with increased cell size. We evaluated macrophage size in these subpopulations using a semiquantitative scoring system and then by quantitative evaluation of macrophage diameter with both showing significant increases in SHR macrophages (P<0.01 for both). To test the extent of IBA1 immunostaining in lung high resolution images were quantified. SHR lungs had more IBA1 immunostaining than WKY (P<0.05). Interestingly, IBA1 immunostaining in small, quiescent macrophages was often homogenous throughout the cytoplasm whereas macrophages that were larger in size often had a central zone of cytoplasmic pallor with stronger immunostaining near the periphery/membrane of the cell. These findings suggest that quantitative and qualitative features of IBA1 immunostaining may be useful tools to detect differences in the activation state of alveolar macrophages.

Support or Funding Information

This work was supported in part by the NIH
Sex Dependent Role of Activating Transcription Factor 3 (ATF3) Expression In Modulating The Asthmatic Phenotype In An Acute Mouse Model Of Airway Neutrophilia
Authors: Spinelli Amy , Ortiz Nathalie Fuentes , Nicoleau Marvin , Sinha Utkarshna , Caruso Carla R , DiAngelo Susan , Chroneos Zissis , Silveyra Patricia
Published: November 2023

Background

Asthma is a chronic lung disease that affects more than 300 million people worldwide, causing more than 250,000 annual deaths. A sub‐population of severe asthma patients have been identified that are distinguished by their high incidence of airway neutrophilia. Patients presenting with this inflammatory profile tend to be less responsive to corticoid steroid treatment and have a higher mortality rate. Therefore, identifying molecules that promote this excessive neutrophil recruitment is essential for better treatment options for these patients. In this study, we aimed to generate and characterize an acute mouse model of airway neutrophilia that could be used to study this particular asthma phenotype.

Methods

Both male and female C57BL/6J mice were challenged with either filtered air (FA) or ozone (1 or 2ppm for 3h). The asthmatic phenotype was assessed by testing airway hyperresponsiveness (AHR) to methacholine challenge using the Buxco system (Penh) and airway remodeling was characterized using the multi‐photon imaging technique‐second harmonic generation to determine collagen content in challenged airways. The extent of airway inflammation was quantified by cell differential assessment of bronchoalveolar lavage fluid (BALF) and lung tissue was harvested for mRNA expression measurements.

Results

We found that ozone exposure leads to airway neutrophilia and AHR. We also saw that expression levels of the stress response activating transcription factor 3 (ATF3) were enhanced in ozone challenged animals with females showing a higher level of up‐regulation compared to males. Interestingly, global knockout of ATF3 was not protective against airway neutrophilia as ATF3−/− male and female mice were observed to have higher levels of neutrophil BALF counts, and female ATF3−/− mice had higher Penh values as compared to wild‐type ozone challenged mice.

Conclusion

This study presents an acute model of refractory asthma whereby ozone challenge initiates AHR and airway neutrophilia. This study also demonstrates the complex role ATF3 plays in regulating airway neutrophilia whereby up‐regulation of this transcription factor is observed to promote the inflammatory phenotype but loss of this protein enhances this inflammatory response in a sex dependent manner.

Support or Funding Information

The authors also thank Todd Umstead for assistance, training and experiments. Dr. Joanna Floros for use of her ozone equipment. NIH Grant KO1HL133520
Inflammation Response of Phage‐Based Films on Titanium Surface in Vitro
Authors: Sun Yuhua , Feng Bo
Published: November 2023

Phage‐based materials exhibit great potential in the applications of tissue regeneration. However, challenges still exist in the development of this field, such as a good understanding of the immune response to phage‐based biomaterials. The objective of this study was to examine the inflammation response of phage film and phage films chemically grafted peptides in vitro. Three different peptides with different affinity to transforming growth factor β1 (TGF‐β1) were used. Macrophage (RAW 246.7) adhesion, proliferation, apoptosis, polarization and expression levels of pro‐inflammatory cytokines, IFN‐γ, TNF‐α, IL‐6, IL‐1β, and anti‐inflammatory cytokines, IL‐10, IL‐4, TGF‐β1, were evaluated. The results showed that the phage films on Ti surfaces would induce the early apoptosis of the adherent macrophage and reduce the proliferation rate. In late culture period, a small amount of cell aggregation was present, but no foreign body giant cells were found. These films promoted sequential M1 and M2 polarization, as measured by expression of surface markers (CCR7 and CD206) and secretion of cytokines. The third day was the turning point of the polarization with increasing secretion of anti‐inflammatory cytokines, IL‐10 and TGF‐β1, although the ratio of M1 polarization was the greatest on the fifth day. No secretion of IFN‐γ and IL‐4 were detected. Macrophage polarization was primarily co‐regulated by phage films and the secreted factors (IL‐10, TGF‐β1 and TNF‐α). Compared to the control group, phage films downregulated the inflammatory response. In addition, the phage film grafted peptide with greatest affinity to TGF‐β1 induced higher early apoptosis of adherent macrophage and IL‐10 expression. The association with the binding of active TGF‐β1 and adsorption of serum proteins will be further studied in detail. The results of this study provide highly encouraging evidence for the safety of potential application of phage‐based materials in vivo.

Support or Funding Information

Natural Science Foundation of China (31570955)
Gas6 reduces cellular respiration and increases reactive oxygen species in immortalized human first trimester trophoblast cells
Authors: Mejia Camilo A , Appiah Margaret B , Lewis Joshua B , Bikman Benjamin T , Hansen Jason M , Reynolds Paul R , Arroyo Juan A
Published: November 2023

Hypertension and proteinuria are two hallmarks of preeclampsia (PE), an obstetric pathology with an elusive etiology that affects 5–10% of pregnancies. At the cellular level, some indications of preeclampsia are decreased trophoblast invasion and increased anti‐angiogenic and cytokine production during placentation. As a protein, growth arrest‐specific 6 (Gas6) has been widely studied in the prevention of apoptosis and enhanced cell migration and invasion. Gas6 has been found to be increased during PE. Previously, we have demonstrated decreased trophoblast invasion with Gas6 treatment and increased blood pressure and proteinuria when rats are injected with Gas6. The effects of Gas6 can be reversed by inhibiting one of its ligands, AXL. Our objective was to determine the effects of Gas6 cellular respiration and reactive oxygen species (ROS) production in the immortalized human first trimester trophoblast cell line SW71. Cultured cells were treated separated into three groups (n=4): Gas6 (600 ng/mL), Gas6 and AXL inhibitor R428 (600 ng/mL), or control. We determined O2 consumption at 37°C in permeabilized cells using the Oroboros O2K Oxygraph (Innsbruck, Austria) with MiR05 respiration buffer. Respiration was determined by using a substrate‐uncoupler‐inhibitor‐titration protocol examining function of complex I, oxidative phosphorylation, complex II and full electron transport system capacity. ROS generation was assessed using the Muse® Oxidative Stress Kit (EMD Millipore, Billerica, MA) as per manufacturer's instructions. Samples were run on the Muse® benchtop flow cytometer (EMD Millipore, Billerica, MA). Overall, our results demonstrated decreases in cellular respiration when cells were exposed to Gas6 when compared to control. We also observed an increase in ROS when cells were treated with Gas6 when compared to control. Both decreased respiration and increased ROS yield were reversed when cells were treated with the AXL inhibitor R428. These results suggest that the stress‐mediated response to the presence of Gas6, as seen in preeclampsia, could involve metabolic pathways that decrease cellular respiration and increase ROS species production.
Profiling of inflammatory biomarkers in synovial fluid from patients undergoing primary total joint arthroplasty
Authors: Finkler Elissa , Wanderling Christopher , Thorson Chase , Galicia Kevin , Statz Stephen , Fareed Jawed , Hopkinson William
Published: November 2023

Introduction

Synovial fluid is an intra‐articular fluid whose main function is to lubricate joints and decrease the coefficient of friction. Synovial fluid contains various proteins, such as lubricin, which contributes to maintaining joint homeostasis and preserving articulating cartilage. The role of inflammatory biomarkers in degenerative joint disease (DJD) has been well established. Previous reports have suggested that increased levels of biomarkers such as IL‐1b, CRP, and MP‐TP play a role in the mediation of inflammatory responses. It is hypothesized that the levels of these biomarkers may provide additional information on the pathogenesis of DJD. This study is designed to profile synovial fluid samples for these biomarkers.

Materials and Methods

This study consisted of 25 patients undergoing primary total joint arthroplasty (TJA) of the hip or knee for DJD. Procedures were performed by one of four attending orthopaedic arthroplasty surgeons. Patients were consented prior to surgery in accordance with LUMC IRB. At the time of arthrotomy, a sample of synovial fluid was obtained using a syringe and placed into a tube containing calcium citrate for preservation and analysis. Biomarkers including IL‐1b, MP‐TF and CRP were measured using commercially available ELISA methods from R&D laboratories (Minneapolis, MN). Total protein levels were measured by nantrope technology.

Results

Total protein levels ranged from 5–65 mg/ml (mean 18 mg/ml +/− standard deviation 13 mg/ml). IL‐1b levels ranged from 0.04–0.96 pg/ml (0.19pg/ml +/−0.18pg/ml). MP‐TF levels ranged from 0–55 pg/ml (14pg/ml+/−19pg/ml). CRP levels ranged from 0–7 ug/ml (1.5 ug/ml +/− 1.9ug/ml).

Discussion

The results from this study demonstrate that synovial fluid from patients with DJD contains detectable levels of inflammatory biomarkers. The presence of IL‐1b and CRP is highly suggestive of an ongoing inflammatory process which may hold significance in the pathophysiology of DJD whereas MP‐TF levels indicate similar degradation products and is a thrombotic biomarker in the form of microparticles complexing with tissue factor. The wide variation in the levels of these markers may suggest differing degrees of degenerative and inflammatory processes. These levels can be quantified to provide more insight into the role of these mediators in the pathogenesis of DJD.
Pro‐Inflammatory and Anti‐Inflammatory Cytokine Expression in Tnf−/− and WT Mice with Chronic Colitis
Authors: Smith Taylor Mikel , Kozik Ariangela , Nakatsu Cindy H , Jones‐Hall Yava L
Published: November 2023

Inflammatory Bowel Diseases (IBD) affect approximately 3.6 million people all over the world, mainly in industrialized countries in North America and Europe. The pathogenesis of IBD is incompletely understood, but it is thought to be caused by an exaggerated immune response to the intestinal microbiome in genetically susceptible people. Crohn's Disease (CD), a sub‐type of IBD, is characterized by recurring bouts of inflammation within the gastrointestinal tract and production of excessive amounts of many pro‐inflammatory cytokines, including tumor necrosis factor (TNF). Anti‐TNF therapy is a common immunotherapy for patients with CD, however, some patients are either non‐responsive to anti‐TNF treatment or become non‐responsive over time. The cause of the non‐responsiveness is not known, but may be related to variations in cytokine production or the microbiome. In this study, we use the 2,4,6‐trinitrobenzenesulfonic acid (TNBS) colitis model to induce chronic, Crohn's‐like colitis in WT mice and mice with deletion of the Tnf gene (Tnf−/−) over a period of five weeks. Spleen and colon samples were collected five weeks post induction of colitis to analyze systemic and local expression levels, respectively, of several pro‐inflammatory and anti‐inflammatory cytokines. We found that the WT mice had significantly more colitis than the Tnf−/− mice, therefore, we hypothesize that WT mice will have higher local and systemic pro‐inflammatory cytokine expression levels and lower anti‐inflammatory cytokines levels when compared to the Tnf−/− mice and that the cytokine levels will correlate with inflammation and alterations in the microbiome.
Comparative analysis of inflammatory cytokines and growth factors in young and old aged normal individuals
Authors: Rodrigues Sandra Maria , Walborn Amanda , Hoppensteadt Debra , Fareed Jawed , Rondina Matthew
Published: November 2023

Introduction

Human aging is a complex phenomenon, characterized in part by chronic, low‐grade inflammation. This increased inflammation, known as “inflammaging”, is a risk factor for increased morbidity and mortality, as most of the diseases associated of aging involve an inflammatory pathogenesis. Accordingly, there is a need to understand the inflammation associated with aging in order to develop therapies targeting inflammaging that may have benefits in the elderly population.

Specific Aim

To compare the levels of cytokines in young (ages 18–55) and old aged (ages 55–85) normal individuals

Materials and Methods

An Evidence investigator cytokine and growth factor biochip array (RANDOX laboratories Ltd.,) was used to perform simultaneous quantitative detection of multiple analytes namely IL‐1α, IL‐1β, IL‐2, IL‐4, IL‐6, IL‐8, IL‐10, VEGF, TNF‐α, IFN‐γ, EGF, and MCP‐1. The Randox biochip is a solid substrate with multiple specific cytokine antibodies attached at pre‐defined sites on the surface. Each cytokine is bound to its specific antibody and then appropriate enzyme‐labeled secondary antibodies are added. The cytokines are quantified by chemiluminescence using a charge‐coupled device camera and imaging system. The concentration of the analyte present in the sample was calculated from the calibration curve

Results

The levels of cytokines VEGF, TNFα, MCP1 and EGF (p>0.05) were observed to be significantly higher in the old aged normal individuals when compared to that in the young aged normals. Two tailed unpaired t test was used to determine whether there were any significant differences between the mean cytokine levels of the two groups and standard error was used for the error bars.

Conclusion

The elevated levels of cytokines in the old aged patients could be a risk factor for morbidity and mortality. With age the damaged organelles, cells and macromolecules tend to accumulate and form host derived endogenous cell debris. There is also increased activation of coagulation system and immunosenescence on aging. All these may contribute as sources of inflammaging stimuli. Therefore, these cytokines can be used as biomarkers for the diagnosis and disease management of various age related pathogenesis.
Gelsolin, but not mucin, inhibits P. aeruginosa swimming motility in vitro
Authors: Husak Alexander B. , Busch Sydney A. , Robarge Jordyn , Bankers‐Fulbright Jennifer L.
Published: November 2023

Pseudomonas aeruginosa is a ubiquitous, opportunistic pathogen responsible for substantial morbidity and mortality in patients with cystic fibrosis (CF). Following initial colonization of the CF lung, P. aeruginosa changes phenotype and becomes virtually impossible to inhibit, kill, or remove. Thus, understanding and preventing initial P. aeruginosa colonization is of great interest. Our lab has previously demonstrated that apical secretions from a human airway cell line, Calu‐3, inhibit P. aeruginosa swimming motility and biofilm formation in vitro, and that this inhibition is lost if proteins > 50 kDa are removed. However, secretions from Calu‐3 cells lacking the CFTR (CF‐like cells) do not inhibit P. aeruginosa. The goal of this project is to identify the inhibitory protein(s) present in non‐CF apical airway secretions. Using reducing SDS‐PAGE, we identified a band at approximately 90 – 100 kDa that was present in normal apical airway secretions but not CF‐like secretions, suggesting that a protein this size may be our target. We have begun testing readily available candidate proteins – mucin and gelsolin ‐‐ to see if they can replicate the inhibitory activity of non‐CF airway secretions. Mucin had no effect on P. aeruginosa swimming motility or biofilm formation, but gelsolin significantly inhibited swimming motility in a dose‐dependent manner. Interestingly, P. aeruginosa biofilm formation was not inhibited by gelsolin. Thus, gelsolin may be responsible for at least a part of the P. aeruginosa inhibition induced by apical airway secretions.

Support or Funding Information

Funding provided by Augsburg College Undergraduate Research and Graduate Opportunities Office and the Augsburg College Biology Department
Better targeting, better efficiency of Adeno‐associated virus gene transfer in the Central Nervous System for expression of Retromer proteins
Authors: Porter Demisha D.L , Jackson Kasey L , Dayton Robert D , Klein Ronald L
Published: November 2023

Researchers are using AAV gene transfer more and more in their neuroscience studies, including optogenetics, cre‐lox targeting, and CRISPR gene editing. Our lab has been using the AAV9 vector serotype to express genes on a wide‐scale basis throughout the central nervous system in order to study a disease that affects cells throughout the central nervous system, amyotrophic lateral sclerosis. This current project explored ways to achieve more efficient expression and in a more targeted manner than our previous work. The recently described AAV PHP.B serotype was administered to rats for the first time, and it did result in significantly more green fluorescent protein expression than did the AAV9 vector in a systematic comparison. In studies comparing the neuron‐specific synapsin promoter with our previous cytomegalovirus/ chicken β‐actin promoter, the latter promoter was noticeably more efficient yet the synapsin promoter did yield a transduction pattern with greater neuron‐selectivity, which was the goal. We also explored intracerebroventricular injections to achieve wide‐scale transduction and better avoid unwanted transduction of peripheral organs. We are combining the synapsin promoter in AAV PHP.B vectors to take advantage of these efficiency and targeting features. Our specific genes of interest that we wish to express are called the retromer proteins. Retromers make up a protein complex that is associated with the trafficking of cellular vesicles through the endosome and the trans‐Golgi network. Viral constructs were made for epitope tagged versions of the retromers VPS29 and VPS35. We hypothesize that the retromers can act to prevent the formation of beta‐amyloid plaques in Alzheimer's disease by altering the trafficking of amyloid precursor protein.

Support or Funding Information

Dr. Ronald Klein, Kasey Jackson, Robbie Dayton, Fidelity Biosciences Research Initiative, Karyopharm Therapeutics, Inc., ALS Association, Meira GTx, The LSU Health Sciences Center Foundation in Shreveport, The American Society for Pharmacology and Experimental Therapeutics, The Society for Toxicology
Surface area may be a more useful risk factor for cerebral aneurysm rupture than maximum diameter
Authors: Fukuda Shunichi , Shimogona Yuji
Published: November 2023

Introduction

The maximum diameter of unruptured cerebral aneurysms is the most significant risk factor of aneurysm rupture. The larger the aneurysm, the easier it is to rupture, and a size of 7 mm or more is clinically significant. This is thought to be at least partially due to hemodynamic involvement. We show here associations between hemodynamic factors and maximum diameter and surface area of cerebral aneurysms by regression analysis.

Methods

We are conducting a multi‐institutional prospective observational clinical study to examine hemodynamics of human aneurysm development, enlargement, and rupture using computational fluid dynamics (CFD); CFD ABO Study. Three‐dimensional CT angiographic images and flow velocities were acquired from 74 patients in the Study.

Results

WSS, NWSS, and WSSG, which are metrics for wall shear stress, at the maximum diameter of 7 mm or more and the surface area of 40 mm2 or more are significantly lower than these at the maximum diameter less than 7 mm and the surface area less than 40 mm2, respectively. Similarly, OSI, GON, and NtransWSS, which are metrics for disturbed flow, at the formers are significantly higher than those at the latters. In addition, the associations between surface area and NWSS, WSSG, GON, or NtransWSS are significant (regression coefficient, 1.548, 1.933, −0.0188, −0.0184, respectively, 95%CI, 0.047 – 3.048, 0.205 – 3.662, −0.033 – −0.0045, −0.035 – −0.021, respectively, P<0.05), but the associations between them and maximum diameter are not at all (Table).

Conclusions

Lower shear stress and stronger disturbed flow are reported to be risk factors for aneurysm rupture. The data by these regression analyses suggest that the surface area is a more useful risk factor for cerebral aneurysm rupture than the maximum diameter.

Support or Funding Information

This study is supported by the Grant‐in‐Aid for the Japanese National Hospital Organization Multi‐Center Clinical Research and AME‐CREST, AMED.
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 Information

Children'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 Information

Funding 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 Information

This 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 Information

This 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 Information

NIH U01 AA018279 (MAC) and NIH T32 AA013527 (LUMC Alcohol Research Program)
The Dual Effect of HIV‐1 Tat and Methamphetamine on Dopamine Transporter Function
Authors: Appadoo Cathaerina Nithchel , Sambo Danielle , Alonge Taiwo , Harvey Brandon , Khoshbouei Habibeh
Published: November 2023

Human immunodeficiency virus (HIV) infected individuals often display cognitive deficits associated with changes in dopamine neurotransmission in the brain. These effects are believed to be partially due to the effects of the HIV trans‐activating regulator protein Tat. Tat is composed of 86 to 101 amino acids and has been previously described to dysregulate the dopaminergic system in the brain. Previous studies indicate that tat targets the dopamine transporter (DAT), a membrane protein that regulates dopamine levels in the brain by clearing released dopamine via a reuptake mechanism. Currently, it is unclear whether Tat‐86 and Tat‐101 similarly or differentially affects DAT activity. DAT is also the primary target for the drug of abuse methamphetamine (METH). METH increases dopamine levels in the brain by altering DAT function, leading to both the rewarding and addictive effects of METH. Importantly, there is a high prevalence of METH abuse in HIV+ patients, although the influence of this comorbidity on dopamine neurotransmission is unknown. In this study, we investigated the dual effects of Tat‐86 and Tat‐101 with METH on substrate uptake via DAT. Using IDT‐307, a specific fluorescent substrate for DAT, we measured IDT‐307 uptake in DAT‐expressing cells exposed to Tat‐86 (30 minutes, 100 nM), Tat‐101 (30 min, 100 nM), in the presence or absence of METH (10 μM). Treatment with Tat‐86, Tat‐101, and METH alone decreased DAT‐mediated uptake. Combined presence of Tat‐86 or 101 and METH augmented the effect of METH, decreasing uptake significantly below baseline. These findings suggest that both forms of HIV‐Tat decrease substrate uptake potentially leading to altered dopamine neurotransmission in the brain and that METH use in HIV infected individuals may further exacerbate the deleterious effects of METH on dopamine neurotransmission. Future studies will investigate whether HIV‐Tat affects DAT‐mediated uptake via increasing DAT internalization from the membrane or altering the conformation of the transporter.
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 Information

NIH 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 Information

NIH grant DA020041 (SGA)
Effects of Juvenile Methylphenidate on Long‐Term Retention in Adulthood
Authors: McWaters Mercedes , Anderson Eden , Matuszewich Leslie
Published: November 2023

Methylphenidate (MPH), or Ritalin, is a commonly prescribed psychostimulant for the alleviation of symptoms in children diagnosed with Attention Deficit Hyperactivity Disorder (ADHD; Centers for Disease Control and Prevention, 2011). Previous research has demonstrated the therapeutic benefits of MPH in the short‐term, but research on the long‐term effects after chronic juvenile exposure is limited. One potential long‐term effect is the alteration of performance on learning and memory tasks, such as the Morris water maze. Prior research in our lab has shown that chronic juvenile MPH treatment enhances the retention of the hidden platform location in the water maze on the first trial of acquisition across training days and MPH‐treated female rats have the greatest improvement across trials within the first day of training. Based on these findings, we hypothesized that MPH may be affecting males' and females' learning and memory differently following juvenile exposure to MPH. Therefore, the purpose of this study was to further investigate the effects of chronic juvenile MPH on long‐term retention in a modified water maze task in male and female rats. This modified task allowed us to explore the potential juvenile treatment and sex differences on short‐term and long‐term memory consolidation. Juvenile rats received an oral dose of 2.0 mg/kg MPH or vehicle on a vanilla wafer from postnatal day 20–34. In adulthood, rats received three sequential training trials to locate a hidden platform in the water maze and then received an acute oral 2.0 mg/kg MPH dose. Short‐term memory was assessed two hours after an acute MPH dose and long‐term memory was assessed 24 hours after acute MPH. Overall, rats showed decreased latencies and distances to find the hidden platform across training, short‐term, and long‐term memory trials, suggesting that all rats learned the task regardless of juvenile treatment. There was a sex by juvenile treatment interaction for latency and distance to find the platform on the long‐term memory trial, suggesting that previous MPH treatment enhanced performance in female rats, but not male rats. To assess retention of the memory of the hidden platform location, rats received a single retention trial once a week for five weeks. All rats continued to learn/reconsolidate the memory of the hidden platform across weeks, irrespective of sex or juvenile treatment. Overall, these results suggest that juvenile MPH may be facilitating females' memory consolidation, as assessed by the long‐term memory trial. These data support our previous findings that female rats may be more sensitive to the long‐term effects of juvenile MPH treatment than male rats.
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 Information

Eleusis PBC