bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulator of G‐protein Signaling 6 (RGS6) Modulates Dopamine D2 Autoreceptor Function in Mice
Authors: Luo Zili , Ahlers‐Dannen Katelin E. , Yang Jianqi , Stevens Hanna E. , Narayanan Nandakumar S. , Fisher Rory A. 
Published: June 2021

The dopaminergic system plays a critical role in the regulation of movement, motivation and cognition and its dysfunction is linked to various neuropsychiatric disorders including Parkinson's Disease. Presynaptic dopamine D2 autoreceptors modulate dopamine signaling in the brain by attenuating its further synthesis and release following the initial synaptic release of dopamine. Consistent with this function, previous studies have demonstrated that pharmacological activation of D2 autoreceptors by quinpirole inhibits presynaptic dopamine release and suppresses locomotion, effects not observed in DAT‐Cre; D2Rfl/fl mice that lack the D2 autoreceptor. RGS6 is a member of the RGS protein family that functions as a GTPase‐activating protein for Gai/o subunits, thereby controlling the intensity and duration of signaling through Gai/o‐linked GPCRs like the D2R. Interestingly, RGS6 is exclusively expressed in dopaminergic neurons of the substantia nigra pars compacta (SNc) of mice and humans, whose loss causes Parkinson's disease, and RGS6 deficient mice exhibit an age‐dependent loss of SNc and striatal dopamine content as well as Parkinson‐like motor dysfunction. Therefore, we hypothesized that SNc dopamine neuron loss in RGS6 deficient mice might result from a lifetime of exaggerated pre‐synaptic D2 autoreceptor signaling culminating in the accumulation of cytotoxic dopamine. To test this hypothesis, we assessed D2 autoreceptor function both in vitro using mouse striatal slices and in vivo. First, we compared the ability of quinpirole to reduce forskolin‐stimulated PKA‐mediated phosphorylation of tyrosine hydroxylase (Ser40) in acute striatal slices derived from WT and RGS6−/− mice. RGS6 deficiency led to exaggerated quinpirole‐mediated inhibition of tyrosine hydroxylase phosphorylation compared to that observed in WT striatal slices. Consistent with these in vitro findings we also found that when 3 mo. WT and RGS6−/− mice, which do not yet display differences in dopamine content, were treated with quinpirole that RGS6−/− mice were more susceptible to quinpirole‐induced suppression of locomotion compared to WT animals despite the mild hyperactivity that has been reported in young RGS6−/− mice. Furthermore, quinpirole also suppressed unsupported rearing in RGS6−/− mice but not WT mice. Together these results demonstrate that RGS6 functions as a critical regulator of D2 autoreceptors in SNc dopamine neurons to modulate dopamine‐dependent motor behaviors. Therefore, loss of RGS6 would be expected to promote hyperactive D2 autoreceptor signaling in these neurons which may underlie their loss in both RGS6−/− mice and humans with Parkinson's.Support or Funding InformationNIH CA161882, MJ Fox 11551, Iowa Cardiovascular Interdisciplinary Research Fellowship T32HL007121.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification/Optimization of Novel Small Molecule Inhibitors of Zika Virus Infection
Authors: Khachatoorian Ronik , Micewicz Ewa D. , Buzzanco Anthony S. , Riahi Rana , Jin Daphne , Ruchala Piotr , French Samuel W. 
Published: June 2021

The rate of Zika virus (ZIKV) infection has been increasing since the original 2014 re‐emergence of the virus in Brazil, and the World Health Organization has warned against potential ZIKV epidemics. Furthermore, ZIKV, a flavivirus family member, has been identified as the cause of microcephaly and other developmental neuroimmunopathologies in fetuses of pregnant women and Guillain‐Barre Syndrome in adults, and to date, no therapeutics or vaccines are available against ZIKV infection. In this study, we screened a limited proprietary library of diverse organic small molecules, and identified a number of leads with significant antiviral activity. Briefly, the anti‐ZIKV activity of the compounds was assayed through plaque‐forming assays in A549 cells. Subsequently, dose response assays were performed for the active compounds using the same experimental setup. Furthermore, we performed structure/activity relationship (SAR) analyses and managed to improve the anti‐ZIKV activity with the most active analogs showing activity in low picomolar range. Finally, we constructed a general model of the overall structure of these anti‐ZIKV compounds which is based on the structural similarities of all active hits. These results identify excellent template(s) with potential for further optimization of antiviral activity through rigorous SAR analyses which may be utilized for the development of inexpensive and orally available anti‐ZIKV drugs. These compounds may also be beneficial for treatment of other viruses in the same family including Dengue virus.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of Prodrug Approaches for Long‐Acting Nanoformulations of Emtricitabine‐Based Regimens
Authors: Al‐khouja Amer , Hobson James J. , Henriquez Stephanie , Meyers David , Curley Paul , Siccardi Marco , Owen Andrew , Flexner Charles , Rannard Steven , Freel Meyers Caren L. 
Published: June 2021

Most current antiretroviral (ARV) medications for HIV treatment and prevention necessitate lifelong daily dosing, and require high levels of patient adherence to be effective. Long‐acting injectable (LAI) ARVs would allow less frequent administration, improving adherence. Technologies currently used to produce LAI‐ARVs are incompatible with the nucleos(t)ide reverse transcriptase inhibitors (NRTIs) due to their high water solubility. It is critical to overcome this challenge to development of LAI NRTI strategies as most current ARV combination therapies include NRTI backbone therapies. We synthesized a series of carbamate‐based emtricitabine (FTC) prodrugs to increase hydrophobicity and improve compatibility for solid drug nanoparticle (SDN) formation. Prodrug activation kinetics were assessed in vitro via HPLC in human muscle, plasma, and liver fractions. Prodrugs were then screened for SDN compatibility using an emulsion templated/freeze drying method. Hits from these screens are defined by the following properties: Z‐average diameter <1000 nm, a polydispersity index of <0.5, and the ability to disperse in water at a concentration of 1 mg/mL. Prodrug release from SDNs and subsequent enzymatic conversion to FTC was quantified by HPLC in human muscle fractions in vitro. Carbamates bearing longer alkyl chains (C7 and C8) were most efficiently activated in all conditions. Hydrolysis rates in liver were higher than those in plasma or muscle.Carbonate moieties at the 5′‐position were hydrolyzed more efficiently than the carbamate moiety under all conditions. Nanoparticle screening at 10 wt% prodrug demonstrated a correlation between calculated logP and the number of hits obtained for carbamate/carbonate prodrugs (39/42), but not for carbamate‐only prodrugs (4/42). Formation of nanoparticles with higher drug loadings was also demonstrated with a hit for an octyl carbamate/carbonate FTC prodrug SDN achieved at 70 wt%. Assessments of in vitro prodrug release from SDNs monitoring FTC formation over time suggested that prodrug release is rate‐limiting under conditions of high SDN concentration. Taken together, this work suggests that carbamate‐based prodrug approaches offer a promising starting point for development of LAI FTC toward a complete LAI‐ARV regimen to improve adherence.Support or Funding InformationNIH 1F31AI129549NIH 5R01AI114405This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lansoprazole inhibits adipocyte differentiation via LXR‐independent mechanism
Authors: Benchamana Ameena , Soodvilai Sunhapas 
Published: June 2021

Liver X receptor (LXR), a ligand‐activated transcription factor, belongs to the nuclear receptor superfamily. It is believed to regulate adipocyte differentiation and lipid metabolism. Lansoprazole (LPZ), an effective FDA‐approved drug used in acid relate disorders, recently is known to be LXR agonist. However, the effect of LPZ on adipocyte differentiation is still unknown. In this study, we investigated anti‐adipogenesis effect of LPZ in 3T3‐L1 pre‐adipocyte and elucidated its possible underlying mechanisms. To determine whether LPZ inhibit adipogenesis, 3T3‐L1 pre‐adipocyte was treated with LPZ (10, 50 and 100 μM) for 48 hours during differentiation induction followed by determination of intracellular lipid accumulation by Oil Red O staining. Our findings showed that LPZ significantly reduced intracellular lipid accumulation only at dose 100 μM without cytotoxicity effect. Co‐treatment the cells with LPZ and LXR antagonists (fenofibrate 10 μM and SR9238 1 μM) for 24 hours failed to attenuate the effect of LPZ on adipocyte differentiation. This result indicated that LPZ inhibited adipocyte differentiation via LXR‐independent mechanism. Expressions of adipocyte differentiation related proteins; peroxisome proliferator‐activated receptor‐γ (PPARγ), CCAAT‐enhancer‐binding protein‐α (C/EBPα), and fatty acid synthase (FAS) were significantly reduced following LPZ treatment. Taken together, our results demonstrated that LPZ inhibits adipocyte differentiation through downregulate PPARγ, C/EBPα, and FAS expression and this inhibition was LXR‐independent pathway. Our results point towards the new indication of LPZ as an alternative therapeutic agent for obesity treatment.Support or Funding InformationThis work was carried out by the support from the Office of the Higher Education Commission of Thailand (Grant to Ameena Benchamana).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of c‐Cbl Antagonists to Promote Corneal Epithelial Wound Healing
Authors: Ceresa Brian 
Published: June 2021

Ligand mediated desensitization is a key negative regulator of cell surface receptors. In the human cornea, epidermal growth factor receptor (EGFR) desensitization, due to the relatively high levels of endogenous EGF in the tear fluid, limits the repair and restoration of the corneal epithelium. The E3 ubiquitin ligase, c‐Cbl is a critical modulator of EGFR desensitization, as it mediates the ubiquitylation of the activated receptor and targets it to the lysosome for degradation. Ultimately, this terminates receptor signaling. We have previously demonstrated that knock down of c‐Cbl results in an enhancement of EGFR‐mediated cell migration that is necessary for healing of corneal epithelial wounds. The goal of these experiments was to identify novel compounds that antagonize c‐Cbl activity and could be developed to enhance EGFR‐mediated corneal epithelial wound healing. Using the published crystal structures of the EGFR:c‐Cbl complex, we identified the point of interaction between the two proteins. We subsequently performed an in silico screen of ~25,000,000 compounds (from the ZINC15 library) to identify compounds that are predicted to antagonize c‐Cbl binding to and ubiquitylation of the EGFR. The top 50 candidates were screened using a biochemical assay for their ability to bind c‐Cbl using Differential Scanning Fluorimetry (Thermofluor™). From that screen, six compounds were identified as c‐Cbl binders and were tested in immortalized corneal epithelial cells to determine which compounds could inhibit c‐Cbl function. Two of the six compounds blocked EGF‐mediated EGFR ubiquitylation, albeit at relatively high concentrations (IC50 were ~50 μM and ~5 μM). Further, these compounds exhibited some cytotoxicity at 100 μM. These compounds represent two lead compounds that can be chemically optimized to enhance their affinity for c‐Cbl and become more potent inhibitors of c‐Cbl activity. The long‐term goal is to use these compounds as therapeutic agents to promote the restoration of damaged corneal epithelium, to minimize patient discomfort, reduce ocular infections, and minimize the likelihood of blindness.Support or Funding InformationResearch was supported by NIH/NEI grant EY027032.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Predicting Resistance To Investigational Microbicide MIV‐150 Using Structure‐based methods and Fluorescence Enzyme Inhibition
Authors: Muwonge Alecia Nicole , Azeem Syeda Maryam , Frey Kathleen M. 
Published: June 2021

Microbicides may be an alternative option in HIV prevention for women affected in sub‐Saharan Africa. Many microbicides are non‐nucleoside reverse transcriptase inhibitors (NNRTIs) that target HIV reverse transcriptase (RT). Resistance mutations result in NNRTI treatment failure. These mutations may also affect the efficacy of chemically similar NNRTIs used as microbicides. The objective of this study was to predict mutations in RT that may cause resistance to investigational microbicide NNRTI MIV‐150. A computational, structure‐based approach was used to predict resistance mutations for MIV‐150. This method calculates changes in affinity (ΔA) and stability (ΔS) imparted by the mutation. The ΔA + ΔS values for K101P predict that this mutation confers high‐level resistance to MIV‐150. Based on this prediction, we analyzed the models and used Molecular Dynamics (MD) to compare the interactions of MIV‐150 with RT wild type (WT) and with RT (K101P). From MD, we found key interactions were lost with K101P. To experimentally validate our findings, we conducted a fluorescence‐based reverse transcription assay for MIV‐150 with RT (WT) and RT (K101P). Our IC50 values showed a 170‐fold change in potency. The assays results validate that the K101P mutation confers resistance to MIV‐150. Our results are consistent with antiviral data reported in the literature. From these results, we believe that this approach is effective for predicting resistance to microbicide NNRTIs.Support or Funding InformationResearch is supported by Dr. Kathleen Frey at Long Island University, Brooklyn.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of Inert, Injectable Sustained Release Formulation for Recombinant Human TIMP‐2 in peptide hydrogels
Authors: Chowdhury Ananda , Yamada Yuji , Schneider Joel P. , Stetler‐Stevenson William G. 
Published: June 2021

Tissue Inhibitor of Metalloprotease 2 (TIMP‐2), is a soluble endogenous 22 kDa metalloprotease inhibitor, that exhibits anti‐tumor, anti‐metastatic and anti‐angiogenic activities both in vitro and in vivo. These effects are mediated via several reported mechanisms including direct MMP inhibition, as well as MMP‐independent processes. Recently we have reported bioprocess development for enhanced production, as well as biochemical and biologic characterization, of recombinant, human TIMP‐2. Thus, allowing pre‐clinical testing of rhTIMP‐2 in multiple murine tumor models. Here we report the development of inert, injectable peptide hydrogel matricies for encapsulation and sustained release of rhTIMP‐2. Additionally, we describe comparison of biophysical attributes and pharmacological properties of rhTIMP‐2 released over time from these biogels.rhTIMP‐2 (with 6X His epitope tag) was produced and characterized as described. Three types of b‐hairpin peptide gels, with varying net charges at physiological pH, exhibited effective encapsulation/retention of up to 4 mg/ml of rhTIMP‐2 (maximal tested concentration). All gels were subjected to oscillatory shear rheology and demonstrate shear‐thin recovery properties post‐incorporation of rhTIMP2. A slow and linear rhTIMP‐2 release profile was obtained from the anionic AcVES3 (net charge: −5) formulation up to 28 days post formulation. Regression equations allow extrapolation of released rhTIMP‐2 based on formulation dose and time elapse. Released rhTIMP‐2 exhibits identical secondary structure (circular dichroism spectra) as well as no detectable change in molecular mass (ESI‐MS) compared with the unformulated rhTIMP‐2. Biogel‐released rhTIMP‐2 inhibits MMP‐2 enzymatic activity and suppresses rhEGF mediated A549 lung cancer cell proliferation in‐vitro similar to unformulated rhTIMP‐2. These findings suggest that these peptide biogels gels could be useful as an injectable vehicle for systemic delivery of rhTIMP‐2 in vivo that would be useful in subsequent dosing experiments studying the anti‐tumorigenic activities of rhTIMP‐2 in murine cancer models.Support or Funding InformationThe authors acknowledge National Institutes of Health Intramural Grants ZIA‐SC‐009179 and ZIA‐BC‐011204 (NCI/CCR Project 8386620) to W.G.S.‐S.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
THE ANTI‐INFLAMMATORY EFFECTS OF ADELMIDROL + SODIUM HYALURONATE (VESSILEN®) IN PRECLINICAL AND CLINICAL STUDIES
Authors: Cuzzocrea Salvatore , Peritore Alessio , Impellizzeri Daniela , Cordaro Marika , Siracusa Rosalba , Fusco Roberta , Gugliandolo Enrico , Crupi Rosalia , Di Paola Rosanna 
Published: June 2021

Interstitial cystitis/painful bladder syndrome (IC/PBS) is a chronic bladder condition characterized by frequent urination, bladder inflammation and pain. It is a particular challenging disease and a clear unmet medical need in terms of identifying new therapeutic strategies. Adelmidrol, the diethanolamide derivative of azelaic acid, represents an effective treatment for human inflammatory disorders. The aim of study was first, to evaluate the anti‐inflammatory effects of intravesical Vessilen® administration (a new formulation of 2% adelmidrol + 0.1% sodium hyaluronate) in acute and chronic animal models of cystitis induced by a single or repetitive intraperitoneal injection of cyclophosphamide (CYP) and then, in patients with IC/PBS treated once weekly by endo‐bladder instillation of Vessilen® for 8 weeks.CYP instillation caused macroscopic and histological bladder alterations, inflammatory infiltrate, increased mast cell number and bladder pain that were reduced by Vessilen® treatment. In addition, an increased expression of nitrotyrosine, a marker of oxidative stress and a decreased expression of endothelial tight junction zonula occludens‐1 were found in CYP‐injected animals. Vessilen® diminished nitrotyrosine and increased zonula occludens‐1 formation. To characterize the inflammatory state and possible reactive oxygen species‐induced pathways involved during chronic cystitis, we also analyzed the nuclear factor‐κB pathway and expression of inducible nitric oxide synthase, interleukin‐1β and monocyte chemoattractant protein‐1. Vessilen® inhibited nuclear factor‐κB pathway and inflammatory mediator levels. Moreover, an increase in nerve growth factor was also found in chronic CYP‐induced cystitis triggering inflammatory response and mechanical hyperalgesia. In that regard, Vessilen® treatment reduced mechanical allodynia and nerve growth factor levels. Interestingly, a reduction in symptom intensity and a significant improvement in the assessment of quality of life were also evident in IC/PBS patients treated with Vessilen®.Thus, in conclusion, we demonstrate that Vessilen® treatment could be a new therapeutic approach in the treatment of human cystitis.Support or Funding Informationno fundingThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
BPC 157 Therapy Heals Tendon Muscle Junction in Rats
Authors: Japjec Mladen , Staresinic Mario , Sebecic Bozidar , Pavlov Katarina Horvat , Drmic Domagoj , Vidovic Tinka , Vlainic Josipa , Seiwerth Sven , Sikiric Predrag 
Published: June 2021

AimAfter tendon was separated from muscle in the right quadriceps muscle in rats, we focused on the tendon‐muscle junction healing and therapy with the stable gastric pentadecapeptide BPC 157. Noteworthy, BPC 157 is an original antiulcer peptide, stable in human gastric juice, used in ulcerative colitis and now multiple sclerosis trials), with particular wound healing ability: healing of transected or crushed muscle, transected or detached tendon, and transected ligament, demonstrated macro/microscopically, biomechanically and functionally (i.e., absent leg contracture), with parenteral and peroral therapy application (Curr Pharm Des. 2017;23(27):4012–4028; J Orthop Res. 2006 May;24(5):1109–17; J Orthop Res. 2006 May;24(5):982–9; J Orthop Res. 2003 Nov;21(6):976–83).MethodsUnder deep rat anesthesia tendon muscle junction of right quadriceps muscle was presented, and without direct transection of tendon muscle junction, the tendon was gently separated from muscle fibres. Medication was BPC 157 (10 ug or 10 ng/kg/day, intraperitoneally or in drinking water (0.16ug or 0.16ng/ml; 12ml/rat/day) while controls received an equivolume of saline (5 ml/kg intraperitoneally) or drinking water only. Macro/microscopical, biomechanical assessment was at 1st, 2nd, 4th, and 6th week after surgery, while functional presentation (walking patterns, leg contracture) was daily assessed.ResultsGross assessment reveals in quadriceps muscle a huge defect between tendon and muscle sustainably progressing throughout the experiment and failed healing. By contrast, in rats underwent tendon muscle junction separation that received BPC 157, the healing was continuously presented. Since very beginning, BPC 157 rats presented defect markedly less and finally grossly reestablished tendon muscle junction (Fig. 1). Also, controls exhibited severe leg contracture, and tottering walk, which were fully counteracted by BPC 157 therapy. These results are completely confirmed by microscopy. Illustratively, at 2nd week controls showed edema of the tendon with moderate lymphocytic infiltrate and formation of granulation tissue, while BPC 157 rats exhibited only minimal lymphocytic infiltrate with no granulation tissue formation in all pentadecapeptide BPC 157 treated animals and with better already reestablished myotendinous junction structures (Fig. 2). Likewise, biomechanic assessment demonstrated only a poor recovery of muscle strength in controls, while a full recovery ensues in BPC 157 treated rats.ConclusionIn addition to the therapy of the injured muscle and tendon, BPC 157 therapy could also specifically benefit the injuries of tendon muscle junction.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.