bluuebunny/crossref_metadata_2025_split huggingface dataset:
mGluR5 regulates ZBTB16 pathway of autophagy in Alzheimer's disease in a sex‐specific manner
Authors: Abd‐Elrahman Khaled S , Hamilton Alison , Ferguson Stephen S. G. 
Published: June 2021

Alzheimer's disease (AD) is characterized by neurotoxicity mediated by the accumulation of beta amyloid (Aβ) oligomers, causing neuronal loss and progressive cognitive decline. We have previously showed that genetic deletion or chronic pharmacological inhibition of mGluR5 by the negative allosteric modulator CTEP, rescued cognitive function and reduced Aβ aggregation in male APPswe/PS1ΔE9 (APPswe) mouse model of AD 1,2. We have recently reported that the novel ZBTB16‐medaited mechanism of autophagy downstream of mGluR5 plays a key role in Huntington's disease (HD) pathology in zQ715 mice 3. In a follow‐up study, we showed that the favorable outcomes of mGluR5 silencing either genetically and pharmacologically in male APPswe were associated with a reduction in ZBTB16 similar to zQ175 mice 4. Here, we show that, unlike male mice, chronic (12 week) treatment with CTEP of female APPswe mice does not improve cognitive function or AD pathology. CTEP treatment did not mitigate memory deficits detected in female APPswe mice during novel object recognition and Morris water maze tasks. We then tested brain samples from male and female APPswe mice following 12‐week treatment with either vehicle or CTEP (2mg/kg) for changes in the autophagic clearance of Aβ oligomers via ZBTB16 pathway. When quantified using ELISA, both male and female APPswe mice exhibited higher level of Aβ oligomers at 6 months of age compared to control. To our surprise, CTEP significantly reduced Aβ burden in male mice yet, it exacerbated Aβ oligomers accumulation in female APPswe mice. mGluR5 blockade in male APPswe mice caused a GSK3β‐dependent degradation of ZBTB16 and stabilization of the autophagy adaptor ATG14. This was paralleled by a reduction in the autophagy marker p62. Interestingly, we did not detect any change in GSK3β phosphorylation, ZBTB16, ATG14 or p62 levels in female APPswe mice following CTEP treatment compared to control. It is worth noting that female APPswe brain also exhibited lower expression of mGluR5 compared to age‐matched APPswe male mice. This study supports the inhibitory role of mGluR5 on autophagy via GSK3β/ZBTB16 pathway that result in impaired clearance of Aβ burden in male APPswe only. We also provide evidence, and for the first time, that some aspects of mGluR5 signaling are not conserved between males and females and there are sex‐specific differences that must be considered when embracing mGluR5 as a potential drug target for neurodegenerative disease.Support or Funding InformationAlberta Innovates Health Solutions and Canadian Institutes for Health ResearchThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
GPR56 is a Novel Platelet Collagen Receptor that Mediates Platelet Activation
Authors: Yeung Jennifer , Rosselli Luciana Kauer , Vizurraga Alexander , Holinstat Michael , Tall Gregory 
Published: June 2021

The adhesion G protein‐coupled receptor (AGPCR) GPR56 (ADGRG1) is required for brain development and nerve myelination. GPR56 is expressed in other tissues, suggesting additional physiological roles. We pharmacologically identified GPR56 in human and mouse platelets, corroborated its presence by immunoblotting, and showed that stimulation of GPR56 with a synthetic peptide‐ or small molecule‐agonist activates platelets. Currently there is an enigma regarding the complete repertoire of collagen receptors that mediate initial platelet interaction with the vessel wound‐exposed collagen sub‐endothelium that fully accounts for platelet activation. GPR56 is a known collagen receptor. We hypothesize that GPR56‐mediated platelet activation is dependent on blood flow shear forced dissociation of the non‐covalently bound GPR56 N‐terminal fragment (NTF) and the C‐terminal fragment (CTF) to expose the GPR56 tethered‐peptide‐agonist. As previously demonstrated, operative dissociation of these GPR56 fragments led to enhanced G13 and Gi activation. Thus, shear force‐induced shedding of the NTF onto collagen is a consequence of GPR56 activation during platelet translocation that leads to platelet shape change and activation via G13 and/or Gi activity. To establish the functional role of GPR56 in platelet activation, human or GPCR deficent (Gpr56−/−) and wildtype (WT) mouse platelets were stimulated with varying concentrations of synthetic GPR56 peptides or 3‐a‐DOG agonist and platelet activation was assessed via a number of tests. Both GPR56 peptide‐ and small molecule‐agonists potently induced platelet aggregation and spreading comparably to the established platelet agonist PAR4 AP. Additionally, dihydromunduletone (DHM), a GPR56 antagonist, inhibited platelet activation mediated by the GPR56 agonists, but not by PAR4 AP. In the case of hemostasis, high shear blood flow rate over collagen coated slides were performed with WT and Gpr56−/−. Gpr56−/− showed decreased platelet adhesion on collagen compared to WT. Gpr56−/− mice also had prolonged hemostatic tail bleeding times compared to the WT. Overall, these functional assays establish GPR56 as an additional collagen receptor that is critical for platelet adhesion and activation in order to maintain hemostasis.Support or Funding InformationNIH RO1 GM120110 (G.G.T), RO1 NS103946 (G.G.T), T32‐HL007853 (CVC), and Michigan Life Sciences FellowshipThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Automated High‐Throughput Drug Discovery in Peptide Hydrogel‐Based 3D Cell Cultures
Authors: Langhans Sigrid A , Worthington Peter , Drake Kathleen , Li Zhiqin , Napper Andrew , Pochan Darrin 
Published: June 2021

Automated cell‐based high‐throughput screening (HTS) is a powerful tool in drug discovery and currently, there is a push to combine complex cell culture systems with HTS to provide more clinically applicable results. Despite the increasing recognition that three‐dimensional (3D) models provide more in vivo‐like cell culture environments, there are mechanistic requirements inherent to HTS as well as material limitations that make this integration for high‐throughput drug discovery challenging. We used the peptide‐based hydrogel MAX8 tagged with the RGDS sequence to create a synthetic extracellular scaffold to culture cells in 3D, and show a preliminary implementation of the scaffold for 3D HTS with a pilot drug screen targeting the pediatric brain cancer medulloblastoma. MAX8 undergoes assembly under physiological conditions and ambient temperature into a hydrogel with a well‐defined, nanofibrillar matrix, desired porosity and stiffness to provide a biocompatible cell culture matrix. Due to the physical gel properties such as shear‐thinning and the fast gelation kinetics, cells can be homogenously encapsulated into evenly dispensed gel‐cell constructs using automatic distribution of cell‐peptide mixtures into 384‐well assay plates with standard liquid handling equipment. 2,202 compounds were screened in the 384‐well format against cells encapsulated in the hydrogel as well as cells growing on traditional 2D plastic. 82 compounds passed the first round of screening at a single point of concentration. 16‐point dose response was done on those 82 compounds, of which 17 compounds validated. Three‐dimensional cell‐based HTS could be a powerful screening tool that allows researchers to finely tune the cell microenvironment getting more clinically applicable data as a result. Here, we have shown the successful integration of a peptide‐based hydrogel into the high‐throughput format.Support or Funding InformationThis work was supported by funds from the NIH IDeA program, with grants from the National Institute of General Medical Sciences NIGMS (P20‐GM103464, P30‐GM114736, and U54‐GM104941), the DO Believe Foundation and the Nemours Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Allotopic Gene Therapy as a Treatment for Mitochondrial Disease
Authors: Paul Jeremiah , Palladino Michael J 
Published: June 2021

Mitochondrial encephalomyopathies (MEs) result from mutations in mitochondrial genes and have been implicated in the aging process as well as several neurological and neuromuscular diseases. Developing genetic methods to knock‐down (KD) or express (rescue) endogenous mitochondrial genes would enable the development of novel therapies for numerous diseases such as neurodegenerative diseases and mitochondrial disorders, including primary encephalomyopathies such as NARP, MLS, and FBSN.We have shown that RNAs can be targeting to mitochondria including long RNAs capable of coding proteins. Our preliminary data suggest these can be expressed by mitoribosomes but expression appears to be inefficient. We hypothesize that removing 5′UTRs and the targeting elements will dramatically improve the efficiency of mitoribosome translation. One approach we are testing is to engineer mitochondrial‐targeted RNA endonucleases (mtREs); RNA endonuclease, which don't exist in nature, could increase the efficiency of translation in the mitochondria by removing interfering 5′ ribonucleotides. We are testing this hypothesis by engineering clones for studies in HELA cells and transgenic animals for studies in Drosophila. We will examine expression efficiency using confocal microscopy and Western blotting.Support or Funding InformationUniversity of Pittsburgh School of Medicine, Summer Undergraduate Research Program (SURP), Molecular Pharmacology Department, Summer 2018 American Society of Pharmacology and Experimental TherapeuticsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of adiponectin‐expressing cells in thymus
Authors: Zhang Yiwei , Cao Handi , Xu Aimin , Wang Yu 
Published: June 2021

ObjectiveAdiponectin, a protein considered as the most abundant adipokine, was found to be expressed in the thymus of wild type mice. The present study characterized the identity and function of adiponectin‐expressing cells in thymus.MethodsThymus was collected from mice with (WT) or without (AKO) adiponectin for isolation and enrichment of thymic nurse cell complexes (TNCs). Different populations of lymphocytes were analyzed by flow cytometry and confocal microscopy. Protein expressions were evaluated by immunofluorescence staining and/or Western blotting.ResultsAdiponectin‐expressing cells were present in TNCs, the specific epithelial cell complexes which engulf thymocytes and provide microenvironment for T lymphocyte development. Compared with age‐matched WT mice, the number and size of TNCs in thymus of AKO mice were significantly reduced. The percentage composition of CD3+ double positive (DP) lymphocytes was significantly decreased, whereas the total amount of double negative (DN) thymocytes significantly increased in TNCs of AKO mice. Adiponectin‐expressing cells were derived from a subpopulation of early T progenitors, which facilitated the formation and function of TNCs. Treatment with adiponectin in vivo or ex vivo could promote the formation and functions of TNCs.ConclusionsAdiponectin‐expressing cells play important roles in the positive and negative selection process of TNCs, thus regulating T‐cell maturation and development in thymus. The results have important implications on the anti‐inflammatory, anti‐diabetic and anti‐tumorigenic properties of adiponectin.Support or Funding InformationThis work was supported in part by grants from Seeding Funds for basic research of the University of Hong Kong, Research Council Grants (17153016) and Hong Kong Health and Medical Research Fund (04151796), Collaborative Research Fund of Hong Kong (C7055‐14G) and the National Basic Research Program of China (973 Program 2015CB553603).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sex‐Specific Differences of Steroid Receptors Following Exposure to Environmentally Relevant Concentrations of Phenothiazine in Fundulus heteroclitus
Authors: Chiari John B , Laperche Jacob M , Patel Roshni , March Nicole , Calvitto Gabriella , Marek Sarah , McGinnis Courtney L. 
Published: June 2021

Agricultural runoff and industrial discharge are known to contaminate ground and surface waters and are not typically removed during water purification. These compounds may have significant effects on biological systems through the disruption of normal endocrine function. This study aims to determine the sex specific effects of phenothiazine (PTZ), a potential endocrine disruptor, on markers for endocrine disruption and stress in Fundulus heteroclitus. The following steroid hormone receptors were assessed: glucocorticoid receptor (GR), androgen receptor (AR), progesterone receptor (PR), estrogen related receptor alpha (ERα), estrogen receptor beta A (ERβA), and estrogen receptor beta B (ERβB). Phenothiazine was detected at levels between 1–2ppm via gas chromatography‐mass spectrophotometry (GC‐MS) in river water sampling conducted in southern Connecticut, but the biological impact of the compound is unknown. Phenothiazine has been used previously as an antipsychotic medication, as well as, industrially in plastics to add softness and flexibility. To assess the impact of phenothiazine, female and male fish were exposed to environmentally relevant concentrations of phenothiazine for one week and sacrificed. Brain, liver, and gonad tissues were removed and pooled from each treatment group. Quantitative PCR (qPCR) was conducted on tissues and analysis showed significant changes in mRNA expression and both a sex‐specific and tissue specific response. There were no changes in the testis and the female liver. In male brain there was a decrease in GR at 1ppm (p<0.05), and an increase in ERβB at 2ppm (p<0.01); in comparison to female brain which had an increase in PR (p< 0.01), AR (p<0.05), ERα (p< 0.0001) and ERβB (p<0.05). In male liver there was an upregulation in GR (p<0.05), and AR (p<0.05) mRNA expression and a down regulation of ERβB (p<0.0001). Lastly, within the ovary there is an increase in AR (p<0.05) and ERβA (p< 0.01) mRNA expression and a decrease in GR (p<0.05) mRNA expression. These findings indicate that phenothiazine may alter endocrine function through the steroid hormone receptors. Collectively, these tissue specific results demonstrate how phenothiazine disrupts normal fish physiology. Additionally, serum cortisol levels were assayed via enzyme linked immunosorbent assay (ELISA) and no significant changes in cortisol levels following phenothiazine treatment were observed.Support or Funding InformationConnecticut Community Foundation, Quinnipiac River FundThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NG291: A Stable B2 Kinin Receptor Agonist that Increases the Permeability of Normal Blood Brain Barrier: Possible Role in Organophosphate Poisoning Treatment
Authors: Rodriguez Sergio Rafael , Martinez Solianne , Martins Henrique Antonio 
Published: June 2021

Organophosphates are phosphorus containing compounds that inhibit acetylcholinesterase and generate a cholinergic crisis that is fatal if not treated. The classical antidotes prescribed are atropine and pralidoxime. They can protect peripherally, but cannot protect the central nervous system. Pralidoxime is the only FDA approved drug that re‐activates the acetylcholinesterase inhibited by organophosphate. Pralidoxime has limited neuroprotective potential because it is not able to cross the blood brain barrier (BBB) effectively. In order to increase this potential, our group is using a stable B2 kinin agonist NG291. This peptide can transiently open the normal blood brain barrier and increase the pralidoxime delivery to the brain after organophosphate poisoning. Sprague‐Dawley (SD) rats were subjected to NG291 treatment (50μg/Kg & 100μg/Kg) via intravenous (IV) route followed by 2% Evans Blue dye injection. 24 hours after the peptide injection the animals were perfused, and the amount of albumin‐Evans blue that reached the brain was qualitatively analyzed. To verify if the transient opening of blood brain barrier causes increase in the brain water content, controls (saline) and rats injected with the peptide NG291 were euthanized 24 hours after the peptide injection and the percentage of water content was measured. To assure that this transient opening is not causing damage to the brain, control and tests were submitted to analysis by histology using Fluoro Jade to verify the presence of dead neurons and glial fibrillary acidic protein (GFAP) to evaluate the astrocyte activation 72 hours after the injection of NG291. NG291‐treated animals have shown infiltration of Evans Blue to the brain parenchyma and accumulation in the ventricles showing increase of blood brain barrier permeation 24 hours after the injection. The water content of the brain did not increase with NG291 treatment. 72 hours after the treatment the histology using Fluoro Jade and GFAP did not show significant changes when compared to controls. We also determined for how long the BBB remains permeable following NG291 administration. In future studies, this peptide will be used to facilitate the delivery of pralidoxime into the brain and therefore increase the overall effect of this drug. This is done in hope to provide those currently exposed to organophosphates with an immediate treatment strategy.Support or Funding InformationUniversity of Puerto Rico Medical Sciences Campus RCMI‐8G12MD007600 Dr. Pedro Ferchmin and Dr. Vesna Eterovic, Universidad Central del CaribeThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Simultaneous Spatial Mapping of Emtricitabine, Tenofovir, and Efavirenz in Mouse Tissue Using MALDI Mass Spectrometry Imaging
Authors: Seneviratne Herana Kamal , Hamlin Allyson N , Bumpus Namandjé N 
Published: June 2021

Emtricitabine (FTC), tenofovir (TFV; prescribed as the tenofovir disoproxil fumarate prodrug) and efavirenz (EFV) inhibit human immunodeficiency virus (HIV) replication and are currently used in combination for HIV treatment. FTC and TFV are nucleotide analog reverse transcriptase inhibitors whereas EFV is a non‐nucleoside analog. Despite the clinical importance of these drugs as commonly used antiretrovirals, their spatial distributions within tissues including those susceptible to HIV infection are poorly understood. Therefore, in this study, we employed a Matrix‐Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI) method to detect, identify and localize FTC, TFV and EFV in mouse tissues following in vivo dosing. For this study, brain and liver were obtained from mice (n=3) 24 h following oral administration of FTC‐TFV‐EFV. Tissue samples were embedded with OCT medium and 20‐micron tissue sections were utilized for tissue imaging experiments. MALDI matrix α‐cyano‐4‐hydroxycinnamic acid was employed to facilitate the ionization and detection of all three compounds, FTC, TFV and EFV. FTC and EFV were detected as their sodium adducts, [M+Na]+ at m/z 270.0319 ± 5 ppm and 338.0166 ± 5 ppm, respectively; while, TFV yielded its protonated molecular ion, [M+H]+ at m/z 288.0856 ± 5 ppm. When collision‐induced dissociation fragmentation experiments were performed using an FTC‐TFV‐EFV mixture, FTC, TFV and EFV exhibited major fragment ions at m/z 130.0409, 270.0746, and 272.0430 corresponding to C4H5FN3O+, C9H13N5O3P+, and C13H10ClF3N+ structural moieties, respectively. Tissue spatial distribution profiles of FTC, TFV and EFV generated using their corresponding ions (sodium adducts and molecular ions) exhibited differences in their distribution across tissue sections. Interestingly, ion intensity of TFV was greater in liver tissue sections than FTC and EFV. In addition, relative homogeneity in distribution of an endogenous lipid species, PC (16:0/OH) indicated that the distribution patterns observed for above molecules were distinct. In order to correlate the tissue distributions of FTC, TFV, and EFV with their lipophilicities, Log P values were calculated. In conclusion, the above results demonstrate the differential spatial distribution of FTC, TFV and EFV in mouse brain and liver following in vivo dosing. Further, results from this study provide important insights into the disposition of FTC, TFV and EFV.Support or Funding InformationThis work was funded by NIH grants U19AI113127, UM1 AI068613 and R01AI128781This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Vitamin A Metabolome in Healthy and Diseased Human Liver Using LC‐MS/MS Analysis
Authors: Zhong Guo , Kirkwood Jay , Won Kyound‐Jae , Tjota Natalie , Jeong Hyun‐Young , Isoherranen Nina 
Published: June 2021

Retinoids are important endogenous compounds responsible for regulating multiple biological events. Currently, the qualitative and quantitative information about retinoid concentrations in human tissues is limited. This prevents a deeper understanding how retinoid homeostasis is regulated in vivo. In this study the hepatic vitamin A metabolome in 50 normal human liver samples was quantified using novel LC‐MS/MS methods and isotope‐labeled internal standards. Measured retinoids included retinyl palmitate (RP; median, 276 nmols/g; range, 35.1–4,282 nmols/g), retinol (ROL; median, 4.9 nmols/g; range, 0.8–39 nomls/g), all‐trans retinoic acid (atRA; median, 39.8 pmols/g; range, 14.9–670.5 pmols/g), 13‐cis‐RA (median, 8.3 pmols/g; range, 1.4–43.5 pmols/g), 4‐oxo‐atRA (median, 18.3 pmols/g; range, 1.8–376.3 pmols/g) and 4‐oxo‐13‐cis‐RA (median, 13.0 pmols/g; range, 5.3–64.0 pmols/g). Two 4‐oxo‐RA isomers were, for the first time, detected in human tissues and their concentrations were comparable to RA isomers. As 4‐oxo‐atRA also activates retinoic acid receptors (RARs), its presence in human liver suggests that 4‐oxo‐atRA is essential in the regulation of retinoid homeostasis. Additionally, atRA concentration was positively correlated with that of other retinoids in normal livers, but not with the relative mRNA expression of LRAT, ALDH1A1, CYP26A1, RARα and RARβ, all of which are retinoid‐related genes. Finally, the vitamin A metabolome was also measured in livers from subjects with non‐alcoholic fatty liver disease (NAFLD) (steatosis, n=9; nonalcoholic steatohepatitis (NASH), n=13) to explore whether the development of NAFLD is associated with disturbed vitamin A homeostasis. The data showed a decrease of hepatic concentrations of RP, atRA, 13‐cis‐RA, 4‐oxo‐atRA and 4‐oxo‐13‐cis‐RA in NAFLD liver samples when compared to normal liver samples, while the hepatic ROL level remained unchanged, indicating that hepatic vitamin A homeostasis is disrupted in NAFLD.Support or Funding InformationThis work was supported in part by grants from the National Institutes of Health R01 GM111772 and T32 DK 007247.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.