bluuebunny/crossref_metadata_2025 huggingface dataset:
Is the Asymmetric Dimethylarginine (ADMA) the Possible Molecule Driving Endothelial Damage in Young Asymptomatic Patients with Cerebral Small Vessel Disease?
Authors: Cifu’ Adriana , Fabris Martina , Janes Francesco , Pessa Maria Elena , Domenis Rossana , Nilo Anna Carmen , Giacomello Roberta , Gigli Gian Luigi , Curcio Francesco
Published: June 2021

Cerebral small vessel disease (CSVD), detected as white matter hyperintensities (WMH) on brain MRI, could be a distinct pathological entity within cerebrovascular diseases. CSVD has a high prevalence in aging population and in stroke patients. In the last years, the increasing use of MRI has led to more incidental finding also in young people without classical vascular risk factor. Our intent was to investigate different pathways implicated in endothelial dysfunction such as inflammation, autoimmunity, coagulation, nitric oxide pathway, in a well selected, homogeneous population of patients with WMH, without significant vascular risk factors or neurological diseases. We selected 35 consecutive patients with evidence of CSVD features at brain MRI and 35 controls age‐sex matched. We evaluated markers for thrombophilia (antithrombin III, protein C, protein S, fibrinogen, plasminogen activator inhibitor – 1, tissue plasminogen activator, von Willebrand factor, homocysteine, C‐reactive protein) and autoimmunity (ANA, ENA, anti‐phospholipids antibodies, lupus anticoagulant) using diagnostic assays. In addition, we evaluated several markers of endothelial activation and inflammation. Interleukin 6, interleukin 10, E‐selectin, P‐selectin and asymmetrical dimethylarginine (ADMA) were assayed by ELISA. Platelet Activating Factor‐Acetyl Hydrolase and nitric oxide were measured with a colorimetric assay, while vascular cell adhesion molecule‐1 and intercellular adhesion molecule‐1 were assayed by Luminex® assay. Statistical analyses were conducted with SPSS 20 (SPSS Inc., Chicago USA). We did not find any significant difference between patients and controls, except for ADMA. Indeed, patients displayed significantly higher levels of ADMA (71.97 ± 36.49 vs 44.46 ± 29.94, p=0.002) than controls. ADMA levels showed positive correlation with the total Fazekas score (r = 0,304; p = 0,01). ROC curve analysis showed a moderate accuracy in discriminating patients and controls (AUC = 0,70; CI 0,57–0,82; p = 0,004): a cut‐off of 46 ng/ml is associated with 80% sensitivity but limited (54%) specificity. In this very large biomarker study, ADMA results the only endothelial marker significantly associated to WMH. This reinforces the hypothesis of the key role of endothelial dysfunction and nitric oxide pathway in CSVD. Further studies should explore the cause‐effect relationship between ADMA pathway and CSVD in selected groups of subjects.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anchor targeting ligands, inorganic nanocomposites, and loads drug in nanobubbles for renal cell carcinoma immune/chemotherapy
Authors: Chan Ming‐Hsien , Liu Ru‐Shi , Hsiao Michael
Published: June 2021

Background

Nanobubble (NB) possess two regions for loading therapeutic agents, namely, the perfluoropentane core and the external surface. Drug release is controlled when encapsulated inside the NB because the drugs are only delivered when an NB is activated by an external stimulus. For instance, ultrasound‐induced cavitation of NBs causes the formation of 150–300 nm holes in the cell membrane, thereby facilitating drug or gene uptake. NBs are ideal vehicles for delivering a wide range of molecules because they are versatile, easily modified for specific targeting of various acoustically active organs, and exhibit low toxicity.

Design

The aim was to synthesize a stable nanosystem which, enhances ultrasound imaging, has optimal luminescence efficiency and stimulates a conducive, synergistic immune/chemotherapy effect. The theranostic agent chosen was, an upconversion nanoparticles (UCNP) associated‐PD1 (UCNP‐PD1) encapsulated inside a folic acid‐nanobubble (Fa‐NB). The cavity of NB can be loaded with the drug, doxorubicin (DOX). Encapsulation of the system within the Fa‐NB provided stability and active targeting to the tumor cell line. Renal cell carcinomas (RCCs) show a high level of folate receptors. When fa‐NB accumulates around RCCs, we can apply ultrasonic energy to release the UCNP‐PD1 from the NB, and bind with the PD‐L1 on the cancer cells and can be tracked by the fluorescence of UCNP.

Results

This encompasses, a Fa‐NB which selectively targets the folic acid receptor found on RCCs. Embedded inside the Fa‐NB is a hybrid nanoconjugate; composed of, UCNPs conjugated to PD1 and loaded with DOX. The aim was to create a nanoscale reagent which implements a selective, immunotherapy and chemotherapy to organs found deep below the body's surface.

Conclusions

A novelly nanotheranostic hybrid system was assembled for immune/chemotherapeutic anticancer treatment. An effective multistep synthesis required the sequential characterization of each individual counterpart, to determine the optimal conditions for the synthesis of the nanocomposite.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interactions of Aldolase A with ATPase Inhibitory Factor 1 Reprogram Energy Metabolism to Maintain Cancer Stemness
Authors: Chang Yu‐Chan , Hsiao Michael
Published: June 2021

Cancer cells utilize glycolysis for the production of ATP and metabolic reprogramming (Warburg effect), as opposed to differentiated bulk tumor cells relying on glycolysis. Cancer Stem cells (CSCs) showed a distinct metabolic phenotype that, depending on the cancer type, could be highly glycolytic or rely on an OXPHOS‐dependent pathway. Aldolase A (ALDOA) has been regarded as one of the key enzymes involved in glycolysis. Overexpression of ALDOA correlated with cancer proliferation and metastasis, and formed a positive feedback loop with HIF‐1alpha during tumorigenesis. Through mass spectrometry‐based ALDOA interactive proteomic database analysis, we have discovered that ALDOA binds directly to ATPase inhibitory factor 1 (IF1). ATPIF1 is a physiological inhibitor of H+‐ATP synthase that is highly overexpressed in the mitochondria of cancer cells. Overexpression of IF1 results in the inhibition of ATP synthesis by the enzyme and a switch to increased aerobic glycolysis and increased ROS‐mediated signaling related to several features of the oncogenic phenotype. We further identified ATPIF1 could be degraded after binding to ALDOA and this function could be rescued by the addition of proteasome inhibitor. In addition, ATP5 family members were found to be upregulated with or without ALDOA enzymatic activity to maintain cancer stemness phenotypes in various types of cancer cells. The additions of IFA could significantly reverse the ALDOA‐induced cancer stemness in a dose‐dependent manner. Moreover, we observed the inverse RNA and protein expression correlations of ALDOA and ATPIF1 predict significant poor prognosis in many clinical cohorts including gliomas, lung and colon cancers. In conclusions, our results provided evidences that ALDOA binding ATPIF1 induce its degradation to modulate energy reprogramming controls and maintain cancer stemness in human cancer cells. This niche may allow new target‐drug development by aiming at the inhibition of ALDOA/IF1 protein binding to suppress cancer stemness.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Localization of the CYP4A Enzymes that Produce 20‐HETE and the 20‐HETE Receptor in the Brain
Authors: Gonzalez‐Fernandez Ezekiel , Xu Rui , He Xiaochen , Wang Shaoxun , Fan Letao , Coolen Lique , Fan Fan , Roman Richard
Published: June 2021

20‐HETE is an arachidonic acid metabolite that is catalyzed by cytochrome P450 (CYP) enzymes of the 4A and 4F families. 20‐HETE is a potent vasoconstrictor that plays an important role in the regulation of cerebral vascular function. Numerous human studies have found that inactivating mutations in the enzymes that produce 20‐HETE are associated with hypertension, stroke, and dementia. Our group previously reported that the Dahl salt‐sensitive (SS) rat also has decreased 20‐HETE levels and exhibits impaired autoregulation of cerebral blood flow and loss of cognitive function following the development of hypertension. A G‐protein coupled receptor (GPR 75) was recently identified as an effector for 20‐HETE, however, little is known about the sites where 20‐HETE is produced in the brain and where its receptors are located. In this study, we used immunohistochemistry (IHC) to identify neuroanatomical structures and landmarks that express CYP4A enzymes and the GPR 75 receptor in SS and control rats. We found that CYP4A enzymes are expressed in hippocampal and hypothalamic neurons, pial and penetrating arterioles in control rats. GPR 75 is expressed in the vascular smooth muscle cells of pial and penetrating arterioles and possibly pericytes at branch points of cerebral arterioles/capillaries. We also found that GPR 75 levels are higher and CYP4A levels are lower in SS vs. control by RT‐PCR, Western Blot and IHC. These findings are consistent with 20‐HETE's emerging role in the regulation of cerebral vascular diseases and dementia.

Support or Funding Information

This study was supported by grants DK104184 (Roman), HL138685 (Roman), AG050049 (Fan), P20GM104357 (Fan and Roman) from the National Institutes of Health, 16GRNT31200036 (Fan) from American Heart Association and the MD/Ph.D. program/Conway Scholarship (Gonzalez‐Fernandez).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Immunomodulation of Mature Dendritic Cells in Parkinson's Disease: A Novel Role of Intracellular Toll Like Receptors
Authors: Paterniti Irene , Campolo Michela , Cordaro Marika , Casili Giovanna , Lanza Marika , Filippone Alessia , Esposito Emanuela , Cuzzocrea Salvatore
Published: June 2021

Parkinson's disease (PD) is a devastating clinical disorders that is characterized by progressive and selective neuronal injury and cell death. Recent studies have provided accumulating evidence for a significant role of the immune system and neuroinflammation in PD pathogenesis. In particular, neuroinflammation associated to PD is the prerequisite for the maturation of dendritic cells (DC) and their migration to the respective sites in the brain. It has become clear that Toll‐like receptors (TLRs), a major family of pattern recognition receptors could mediate innate and adaptive immune response, providing an important mechanism by which microglia are able to sense both pathogen‐ and host derived ligands within the CNS.

The aim of our study was to evaluate the role of TLR 7,8 and 9 in DC maturation that trigger to adaptive autoimmune response related to PD.

We performed an in vivomodel of PD, by MPTP, in single KO mice for TLR7−, TLR8− and TLR9−; and in double KO mice for TLR 7/8−/−and TLR7/9−/−. All animals was compared with WT animals used as a control group.

Animals were sacrificed after 8 days and their midbrains were harvested, sectioned and processed in order to evaluate: the principal markers of PD with particular attention to TH, DAT and α‐synuclein aggregate; astrocytes and microglia activation through GFAP and IBA‐1; pro‐inflammatory cytokine's expression such as INF‐γ and TNF‐α like major product of activated microglia and astrocytes. Moreover to evaluate the formation of neuromelanin pigments in the midbrain we made the Masson‐Fontana staining.

The result obtained demonstrated that the genetic absence of TLR 7,8 and 9 modified PD pathway increasing significantly the immunoreactivity for TH and DAT compared to PD groups where we found an evident reduction of these markers.

Therefore, TLRs could be considered as a possible target to develop new therapies for neurodegenerative disorders.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
N‐Palmitoylethanolamine‐oxazoline (PEA‐OXA): a new therapeutic strategy to reduce neuroinflammation and oxidative stress associated to vascular dementia
Authors: Cuzzocrea Salvatore , Impellizzeri Daniela , Siracusa Rosalba , Cordaro Marika , Crupi Rosalia , Peritore Alessio Filippo , Gugliandolo Enrico , D'Amico Ramona , Di Paola Rosanna
Published: June 2021

Recent studies revealed that pharmacological modulation of NAE‐hydrolyzing acid amidase (NAAA) can be achieved with PEA oxazoline (PEA‐OXA). Hence, the aim of the present work was to thoroughly evaluate the anti‐inflammatory and neuroprotective effects of PEA‐OXA in an experimental model of vascular dementia (VaD) induced by bilateral carotid arteries occlusion. At 24 h after VaD induction, animals were orally administered with 10 mg/kg of PEA‐OXA daily for 15 days. Brain tissues were handled for histological, immunohistochemical, western blot, and immunofluorescence analysis. PEA‐OXA treatment evidently reduced the histological alterations and neuronal death induced by VaD and additionally improved behavioral deficits. Further, PEA‐OXA decreased GFAP and Iba‐1, markers of astrocytes, and microglia activation, as well as MAP‐2, a marker of neuron development. Moreover, PEA‐OXA reduced oxidative stress, modulated Nrf2‐mediated antioxidant response, and inhibited the apoptotic process.

Some drugs may demonstrate their healing potential by regulating neuroinflammation, rather than by their habitually attributed actions only. Palmitoylethanolamide (PEA) is a prototype ALIAmide, well‐known for its analgesic, anti‐inflammatory, and neuroprotective properties. The inhibition of PEA degradation by targeting NAAA, its catabolic enzyme, is a different approach for treating neuroinflammation. This research offers new insight into the mechanism of PEAOXA‐induced neuroprotection. Thus, the modulation of intracellular NAAA by PEA‐OXA could offer a novel means of controlling neuroinflammatory conditions associated with VaD

Support or Funding Information

no fundings

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
(−)‐α‐Bisabolol Promotes Neuroprotection on Neuronal Damage, Motor and Memory Deficits Induced by Focal Cerebral Ischemia in Mice
Authors: Fernandes Mara Yone Soares Dias , da Silva Ana Thaís Araújo , Bezerra Jessica Rabelo , Pires Rayssa Costa , Mendonça Rafael Costa , de Andrade Geanne Matos
Published: June 2021

Brain ischemia pathophysiology involves a complex cascade of events such as inflammation and oxidative stress that lead to neuronal loss and cognitive deficits. (−)‐α‐Bisabolol is a sesquiterpene alcohol obtained from plants such as Vanillosmopsis erythropappa and Matricaria chamomilla, with anti‐inflammatory and antioxidant properties. The present study aimed to investigate the effects of (−)‐α‐Bisabolol (α‐Bis) on neuronal damage and memory deficits induced by permanent middle cerebral artery occlusion (MCAO). Mice were submitted to MCAO and were treated with α‐Bis (50, 100, and 200mg/kg, orally), 3 hours after MCAO and during 4 days. The parameters studied were: infarcted area, memory deficits, myeloperoxidase (MPO) activity, and immunoreactivity for cytochrome C, NF‐κB, and superoxide dismutase (SOD‐2). α‐Bis treatment reduced significantly cerebral infarction, neurological and aversive memory deficits, decreased MPO, and cytochrome c, NF‐κB and SOD‐2 immunoreactivity when compared with MCAO group. Taken together, our study demonstrated neuroprotective effect of (−)‐α‐Bisabolol against ischemia‐induced cerebral injury in mice. We propose that the neuroprotection offered by (−)‐α‐Bisabolol can be attributed, at least in part, to its anti‐inflammatory activity.

Support or Funding Information

FUNCAP, UFC

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulation of Microglia by P2X and P2Y Receptors
Authors: Campagno Keith , Lu Wennan , Albalawi Farraj , Cenaj Aurora , Tso Huen‐Yee , Mitchell Claire
Published: June 2021

Microglial cells are involved in a complex series of physiologic and pathophysiologic interactions in the nervous system, and the receptors responsible for mediating these responses are central to coordinating these responses. ATP is a key extracellular signaling molecule in brain and retinal injury, and our previous results indicate release of ATP through pannexin hemichannels following mechanical injury to astrocytes. We ask here whether ATP alters migration, reactivation state and cytokine release in retinal and brain microglial cells. Using a Boyden chamber, ATP was shown to be a powerful chemoattractant for primary microglial cells isolated from both the brain and the retina. Detection of Hoechst‐labeled microglia on the lower side of the chamber peaked after 3 hours. Microglial migration was reduced by exposure of the cells to the P2Y12 antagonist ARC‐69931, while the P2X7 antagonist A839977 had no effect. Exposure of primed microglia to ATP led to the release of IL‐1β. This release was mimicked with P2X7‐specific agonist BzATP, and inhibited with antagonist A839977. Preliminary findings suggest ATP increased markers for the M1‐like activation state and decreasing the M2‐like activation state of microglial cells. Together these data suggest that stimulation of P2 receptors for ATP has a key role to play in migration, cytokine release and reactivate state of microglial cells. We speculate that ATP is a key signaling connection between astrocytes and microglia and helps link mechanical strain to inflammatory signaling in neural tissue.

Support or Funding Information

This work is supported by NIH Grants EY015537 and EY013434, and the Vision Research Core Grant EY001583.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mutant Huntingtin‐Calmodulin Interaction: Potential Therapeutic Target for Huntington's Disease
Authors: Kapadia Khushboo , Roy Anuradha , McDonald Peter , Klus Nicholas , Frankowski Kevin , Aubé Jeffrey , Muma Nancy
Published: June 2021

Huntington's disease (HD) is a neurodegenerative disorder caused by an autosomal dominant mutation in the huntingtin (htt) gene. Previous studies in our lab demonstrated that disrupting the binding of mutant huntingtin (mhtt) to calmodulin (CaM) had beneficial effects in cell culture and the R6/2 transgenic animal model. The goal of the current study is to identify and develop small chemical compounds that are non‐toxic and can selectively disrupt the binding of mhtt to CaM. To this end, we employed a high throughput AlphaScreen assay along with counter‐screening assays and identified 481 hit compounds that disrupt the interaction between (His)htt‐CaM(GST). The structures of the hits obtained were analyzed and structurally diverse representative scaffolds were chosen for further development. Analogs of these compounds synthesized from the selected scaffolds were re‐screened in the primary AlphaScreen assay and tested for cytotoxicity. Next, assays were employed to determine if the hits identified in the primary screen can selectively disrupt the mhtt‐CaM interaction without affecting other functions of CaM. The compound selectivity is being determined using two CaM dependent enzymes which are abundantly expressed in neuronal cells and play an important role in neuronal function; Ca+2/CaM dependent death associated protein kinase 1 (DAPK1) and Ca+2/CaM dependent protein kinase 2 gamma (CAMK2ϒ). We have identified numerous compounds that have shown preferential activity in disrupting the (His)htt‐CaM(GST) interaction. Compounds that were non‐toxic and selectively disrupted the binding of mhtt to CaM are tested in cell‐based screening assays to determine if the compounds are protective against deleterious effects of mhtt expression. So far, we have identified two scaffolds that selectively disrupt the binding of mhtt to calmodulin and show protection against mhtt‐induced toxicity. Studies are ongoing to determine if these scaffolds will reduce accumulation of aggregated htt protein and normalize mitochondrial deficits associated with mhtt expression. In the future, these compounds will be tested for their protective effects in animal models of HD. Overall, these studies will aid in identifying compounds that will serve as novel and promising biological probes for drug development in HD.

Support or Funding Information

1RO1NSO88059

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dopamine transporter is dysregulated on the peripheral immune cells of drug naïve Parkinson's Disease patients
Authors: Gopinath Adithya , Mackie Phillip , Saadatpour Leila , Doty Andria , Ramirez‐Zamora Adolfo , Streit Wolfgang J. , Okun Michael , Khoshbouei Habibeh
Published: June 2021

Introduction

Parkinson's Disease (PD) is characterized by loss of substantia nigra (SN) dopaminergic neurons, resulting in reduced CNS dopamine transmission. PD is widely thought to start in the periphery; however, whether and how PD affects peripheral dopamine transmission remains unknown. Peripheral immune cells express key dopaminergic proteins, including dopamine transporter (DAT) and tyrosine hydroxylase (TH). Therefore, it is possible that the peripheral dopamine system is affected by PD pathology.

Objective

We hypothesized that peripheral immune cells of PD patients exhibit dysregulated dopamine homeostasis.

Methods/Results

Using flow cytometry we found human peripheral blood mononuclear cells (PBMCs) constitutively express DAT and TH with 90% of CD14+ cells expressing both markers. Upon examining PBMCs of PD patients receiving a variety of treatments, we found that DAT/TH positive PBMCs are elevated in PD patients compared to healthy controls (n=67 independent biological replicates, P<0.05) irrespective of the treatment modality applied. Importantly, in drug naïve patients diagnosed with PD we observed the highest increase in DAT/TH positive monocytes (n=5, P<0.05) compared to both treated PD patients and healthy individuals, suggesting observed changes correlate with disease pathology and not treatment interventions. Collectively, these data suggest a system‐wide dysregulation of the dopamine system on peripheral immune cells in PD. To further understand the functional consequences of increased DAT+/TH+ PBMCs, we asked if DAT function was altered on PD monocyte‐derived cells. Live cell fluorescence microscopy and biochemical analysis confirmed healthy human monocyte‐derived macrophages (MDM) express membrane‐localized, canonically functional DAT (Km=3.2mM). Surprisingly, relative to healthy age‐matched controls, PD patients' MDMs exhibited dramatically elevated DAT‐mediated substrate uptake and increased membrane localization (p<0.0001, n=5 biological replicates).

Conclusions

Taken together, these data are consistent with the interpretation that in PD, peripheral dopamine homeostasis is dysregulated. Importantly, this presents PBMC DAT/TH as a potential biomarker for PD and suggests the peripheral dopamine system is functionally linked to the CNS dopamine system. Future work will aim to validate this biomarker and investigate the mechanistic connection between peripheral‐CNS dopamine systems to elucidate the pathophysiological role of dysregulated monocyte dopamine transmission in PD.

Support or Funding Information

Mcknight Brain Institute; NINDS 0016811

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dendrobium nobile Lindl. alkaloids alleviate Mn‐induced neurotoxicity via PINK1/Parkinmediated autophagy in SH‐SY5Y cells
Authors: Fu Xiaolong , Wang Xueting , Zeng Ru , Wu Qin , Lu Yuanfu , Shi Jingshan , Zhou Shaoyu
Published: June 2021

Chronic environmental and occupational exposure to manganese (Mn) results in a progressive neurological disorder referred to as manganism, a syndrome closely resembling Parkinson's disease (PD). The loss of dopaminergic neurons is critically involved in pathological progression of Mn‐induced manganism. Identification of molecular modulators of Mn‐induced neurotoxicity is essential to the development of neuroprotective strategies or effective treatments against Mn manganism. Mitochondrial dysfunction has been increasingly appreciated as a key determinant in Mn induced Parkinsonism. It has been known that PD‐associated mitochondrial proteins, PINK1 and parkin, regulate mitochondrial degradation in the development of neurological disorders. This study was designed to determine whether Dendrobium nobile Lindl. alkaloids (DNLA), a traditional Chinese medicinal herb extract, plays protective function over Mn‐induced neurotoxicity, and to investigate whether the modulation of PINK1/Parkin‐mediated autophagy is involved in the mechanism of DNLA‐mediated cell protection in Mn toxicity. The human dopaminergic cell line SH‐SY5Y cells were utilized as an in vitro model of Mn cytotoxicity. It was found that the treatment of the SH‐SY5Y cells with Mn resulted in concentration‐dependent cell death, accompanied by a decrease in mitochondrial respiration capacity, whereas pretreatment of cells with DNLA significantly alleviated cell toxicity induced by Mn and improved mitochondrial function. Analysis of PINK1 and Parkin expression revealed that pretreatment of the cells with DNLA significantly inhibited the Mn‐elicited decrease in the protein expression levels of PINK1 and Parkin. In addition, the Mn‐treated cells exhibited increased expression of LC3‐II and accumulation of p62, which was noticeably reversed by the pretreatment of cells with DNLA. These results demonstrate that DNLA inhibits Mn induced cytotoxicity, which may be mediated through modulating PINK1/Parkin‐mediated autophagic flux and improving mitochondrial function.

Support or Funding Information

This work was supported by grant from the National Natural Science Foundation of China (Grant No. 81460548).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Trans‐synaptic regulation of group III mGluR pharmacology by endogenous allosteric modulators implicated in neuropsychiatric disease
Authors: Dunn Henry A. , Dao Maria , Orlandi Cesare , Zucca Stefano , Martemyanov Kirill A.
Published: June 2021

Functional characterization of the GPCR interactome has focused predominantly on intracellular interacting‐proteins, yet GPCRs are increasingly found in complex with extracellular proteins. Extracellular leucine rich repeat fibronectin type III domain containing 1 (ELFN1) was recently reported to physically anchor mGluR6 and mGluR7 across retinal and hippocampal synapses, respectively; however, the consequence and extent of trans‐synaptic interactions on GPCR physiology and pharmacology had been unknown. We explored the functional implications of ELFN1‐mGluR interactions by developing a novel transcellular GPCR signaling assay platform to monitor changes in mGluR activity in one cell following its exposure to separate ELFN1‐containing cells. Using this platform, we found the first evidence of allosteric modulation of GPCR activity in trans; whereby ELFN1‐expressing cells in co‐culture altered both agonist‐induced and constitutive receptor activity of group III mGluRs expressed in separate cell populations. We further adapted this platform for various readouts and determined transcellular ELFN1‐mediated alterations of group III mGluR cAMP signaling was not a consequence of receptor membrane expression or desensitization, but instead a direct consequence of altered G protein activation as seen by BRET‐based real‐time kinetic assays. Furthermore, we have identified additional trans‐synaptic binding partners of group III mGluRs with previously unknown function and demonstrate selective pharmacological consequence and relevance to neuropsychiatric disease. Our findings demonstrate that accepted principles of group III mGluR pharmacology are critically altered via extracellular interactions with endogenous trans‐synaptic allosteric modulators, and disruption of these interactions may endow susceptibilities to neuropsychiatric disease.

Support or Funding Information

This work was supported by NIH Grants EY018139 and DA026405 (to K.A.M.). H.A.D. is the recipient of a Canadian Institutes of Health Research Postdoctoral Fellowship award.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Establishing the Framework to Evaluate Negative Modulators of Mu Opioid Receptor Signaling in Cells
Authors: Stoveken Hannah M , Wang Dandan , Masuho Ikuo , Grill Brock , Martemyanov Kirill A
Published: June 2021

Opioid addiction and accidental overdose have arisen as critical health issues as the prevalence and availability of this addicting class of drugs have dramatically increased. The mu opioid receptor (MOR) is the primary analgesic mediator in the brain and is activated by the class of drugs known as opioids. MOR is a G protein coupled receptor (GPCR) that signals through the heterotrimeric G protein class Gi/o. Once activated, the heterotrimeric G proteins transduce the opioid signal to other effectors including adenylate cyclase, voltage‐gated calcium channels, and G protein‐coupled inward‐rectifying potassium (GIRK) channels, among others. Together, this signaling renders the cell less excitable and blocks transmission of pain responses but also elicits unwanted side effects. Endogenously, MOR signaling is regulated to terminate or tune signaling and hence to alter the duration or intensity of the opioid response. Regulators of G protein signaling (RGS) proteins, for example, serve as GTPase‐activating proteins to inactivate Gi/o and terminate MOR signaling. We hypothesized that there may be undiscovered negative MOR regulators that could modulate MOR signaling to lessen opioid side effects. We are currently developing an unbiased forward genetic screen in c. elegans to uncover potential new cell‐autonomous MOR modulators. While the screen is an ongoing effort, we are working to establish the framework for evaluating potential hits in cell‐based assays of MOR signaling. As proof of concept, we examined the effect of the known negative MOR regulator R7BP‐RGS7‐Gb5 complex in cellular assays of MOR function and/or trafficking. Overall, this project is aimed at uncovering and determining the molecular mechanisms of potential new anti‐opioid signaling pathways that could be pharmacologically modulated to mitigate negative opioid‐related side effects.

Support or Funding Information

National Institutes of Health R01 to KM

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of CXCR4 signalling and ligand binding by CXCL17
Authors: White Carl W , Pfleger Kevin , Hill Stephen J
Published: June 2021

CXCL17 is the most recently described chemokine ligand and is highly expressed in mucosal tissues. CXCL17 has been associated with innate responses and acts as a chemoattractant for monocytes and macrophages, but has also been shown to play a role in angiogenesis for tumour development [1]. However currently CXCL17 is poorly characterised, in part, as it signals through a yet unidentified receptor. Initial reports suggested CXCL17 may act as an agonist for the GPCR GPR35 [2], however two recent studies failed to replicate this finding [3,4]. The CXCR4 chemokine receptor is highly expressed in many immune cells that respond to CXCL17. We therefore aimed to investigate if CXCL17 modulates CXCR4‐mediated signalling in model HEK293 cells.

Ligand binding and signalling was investigated using NanoBRET assays. For ligand binding HEK293 cells stably expressing Nluc/CXCR4 were incubated for 1 hour with CXCL12‐AF647 (12.5 nM) in the absence or presence of CXCL17 (0.3 nM – 1 μM). For β‐arrestin2 recruitment assays HEK293 cells transiently transfected with CXCR4/Nluc or CXCR4/Rluc8 and β‐arrestin2/Venus were incubated with CXCL17 (0.3 nM – 1 μM) or CXCL12 (10 nM) in the absence or presence of CXCL17 (0.3nM – 1 μM). For G protein activation assays HEK293 cells transiently transfected with Gαi1/Nluc and Venus/Gγ2 were incubated with CXCL17 (1 μM) or CXCL12 (0.1 pM) in the absence or presence of CXCL17 (1 nM – 1 μM). Luminescence was generated by addition of furimazine and changes in BRET following ligand addition was measured between the luciferase and the fluorescent acceptor. Filtered light emissions were measured using a PHERAstar plate reader (BMG).

NanoBRET competition ligand binding studies showed CXCL17 inhibited CXCL12‐AF647 binding to Nluc/CXCR4 in a concentration dependent manner (pKi; 6.33 ± 0.33, n=4). In β‐arrestin2 recruitment experiments, CXCL17 resulted in a decrease in constitutive BRET between CXCR4/Rluc8 and β‐arrestin2/Venus (pIC50; 7.24 ± 0.11, n=3) and also inhibited the increase in BRET mediated by exogenous CXCL12 in a concentration dependent manner (pIC50; 6.97 ± 0.21, n=6). Similarly CXCL17 inhibited CXCL12 mediated G protein activation (pIC50; 6.73 ± 0.62, n=4) as well as reducing constitutive CXCR4 mediated G protein activation. CXCL17 did not appear to have agonist effects at CXCR4.

This work shows for the first time that CXCL17 is a low affinity inhibitor of CXCR4‐CXCL12 ligand binding, and acts as an inverse agonist of constitutive and CXCL12‐induced CXCR4 signalling.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Extracts of Two Relatively Safe South African Alien Weeds have Synergistic Activity with Conventional Antibiotics against some Urinary Tract Pathogens
Authors: Omokhua Aitebiremen Gift , Van Staden Johannes , McGaw Lyndy Joy
Published: June 2021

Plants have potential to play a role in the treatment of urinary tract infections, as they contain important bioactive components. These bioactive components may act in synergism with conventional antibiotics which are presently under threat of microbial resistance. They also play a key role in the reduction of oxidative stress. Synergistic activity of dichloromethane, acetone, 70% ethanol, 50% methanol and hot water extracts of the southern African alien weeds Chromolaena odorata and Tithonia rotundifolia combined with gentamicin and amphotericin B was studied using the checkerboard method. Antioxidant activity of the extracts was determined using the quantitative 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical scavenging and 2,2′‐azinobis‐(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) decolorisation methods. The anti‐inflammatory activity was determined through the 15‐lipoxygense enzyme assay. Toxicity screening of the extracts was carried out against Vero monkey kidney, human carcinoma Caco‐2 and human liver C3A cell lines using a colorimetric assay. Synergistic to additive effects were observed among all tested extracts in combination with the conventional antibiotics against all tested strains which are Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Salmonella Typhimurium, Aspergillus fumigatus and Candida albicans. Although the extracts of both plant species had antioxidant activity, the extracts of C. odorata displayed better activity in the ABTS assay with the 70% ethanol extract showing a 50% inhibitory concentration (IC50) of 1.96 μg/mL, better than that of vitamin C (IC50 = 2.027 μg/mL). The dichloromethane extract of T. rotundifolia had better radical scavenging activity in the DPPH assay with an IC50 of 16.231 μg/mL. All the extracts of C. odorata had anti‐inflammatory activity better than that of the positive control quercetin (IC50 = 24.601 μg/mL) except for the acetone extract which was slightly higher (IC50 = 26.098) than the positive control. Lethal concentrations (LC50) of the extracts against Vero, Caco‐2 and C3A cells ranged from 0.78 to 0.96mg/mL, 0.198 to 0.32mg/mL and 0.67 to 0.88 mg/mL respectively, indicating that the extracts are relatively safe in vitro. Results from this study indicate that the extracts of these weedy plant species contain comparatively non‐cytotoxic bioactive compounds that may act in synergy with conventional antibiotics in the treatment of urinary tract infections. Additionally they may reduce oxidative stress and inflammation. Further studies to determine the active component(s) of these weed plants are ongoing.

Support or Funding Information

No funding

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tangeretin Protects against Ethanol‐Induced Injury in Gastric Mucosa of Rats via its Antioxidants and Anti‐inflammatory Activity
Authors: Arafa El‐Shaimaa A. , Islam Mohammad Waseemul
Published: June 2021

Tangeretin (TG), a polymethoxy citrus flavone, has attracted attention due to its significant antioxidant and anti‐inflammatory activities. It showed valuable pharmacological actions and protected against cancer, diabetic‐, liver‐ and renal‐induced injuries. However, its antiulcer activity has not been investigated.

This study aimed to explore the possible protective effects of TG against ethanol‐induced gastric injury in rats. Adult male Wistar rats were used and divided into five groups: Control, TG, EtOH, EtOH+TG and EtOH+ Pantoprazole (PAN). Gastric injury was induced by a single dose of Absolute ethanol (5ml/Kg, p.0).

Alcohol administration leads to significant macroscopic, histologic and biochemical gastric injuries. Treatment with TG (100mg/kg p.o.) protected against EtOH‐induced gastric injuries as demonstrated by the decrease in the ulcer and bleeding scores. Tangeretin boosted the antioxidants properties of the gastric mucosa via the inhibition of Malondialdehydes (MDA) and nitric oxides (NO) while improving glutathione (GSH), superoxide dismutase (SOD) and the nuclear factor erythroid 2–related factor 2 (Nrf2). Tangeretin curbed gastric inflammation by decreasing myeloperoxidase (MPO) and tumor necrosis factor‐α (TNF‐α) while increasing the anti‐inflammatory interleukin‐10 (IL‐10). In addition, TG caused the upregulation of the gastric cytoprotective prostaglandin E2 (PGE2). These results were comparable to those produced by PAN; the proton pump inhibitor.

These findings reported for the first time the gastroprotective actions of TG in EtOH‐induced gastric injury which were mediated via suppression of inflammation and oxidative stress in addition to enhancing the antioxidant and the cytoprotective defenses.

Support or Funding Information

This work has been financially supported by the research grant# 2017‐A‐PH‐03 from the Deanship of Graduate Studies and Research, Ajman University.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Beneficial effects of aqueous extracts of Mikania glomerata Sprengel and Mikania laevigata Schultz Bip ex Baker on the inflammatory response in rats with acute pancreatitis
Authors: de Souza Pereira Della Pasqua Camila , Antunes Edson , Sawaya Alexandra Christine Helena Frankland , Campos Rafael , Landucci Ellen Cristina Teizem
Published: June 2021

Objectives

According to previous studies, aqueous extracts of Mikania glomerata (MG) and Mikania laevigata (ML) possess considerable anti‐inflammatory activity. Altogether, these findings suggest that these species has a potential effect in the treatment of inflammatory conditions, particularly those involving the gastrointestinal tract. In view of this, it is believed that these species may present important effects in the treatment of acute pancreatitis. Since there is no specific pharmacological therapy for acute pancreatitis, the elucidation of this parameter is the main objective of this study, in an attempt to prove the therapeutic efficacy of these plants under this condition.

Material and Methods

The extracts were characterized by Ultra High Efficiency Liquid Chromatography coupled to Mass Spectrometry (UHPLC‐MS). The animals were treated per oral route (p.o.), through the gavage technique, in the final volume of 500 μL, for 7 consecutive days and then submitted to induction of acute pancreatitis (AP). Male Wistar rats were submitted to injection of sodium tauracholate (30 mg/kg) into the common bile duct. After 4 hours the animals were sacrificed, when blood, pancreas and left lung were collected for analysis of pancreatic edema, pancreatic and pulmonary neutrophil infiltrate (MPO), elevation of serum amylase, TNF‐α in serum and interleukins.

Results and Discussion

Induction of AP by sodium tauracholate significantly increased all the parameters evaluated when compared with the respective sham group. The pretreatment with MG (400 mg/kg) significantly reduced pancreatic oedema, pancreas and lung MPO activity. The same profile of reduction was observed in the pretreatment with coumarin (75 mg/kg). The pretreatment with ML showed a significant reduction for all doses tested in the pancreatic oedema and lung MPO activity. For pancreas MPO activity, the doses of 200 and 400 mg/kg showed significant reduction of this parameter.

Conclusion

Our results showed that aqueous extracts of M. glomerata and M. laevigata decreases inflammation acute pancreatitis, making it of interest in future approaches to treat this condition. However, additional studies are being made by our group to establish the effectiveness of this mechanism.

Support or Funding Information

Camila S. P. Della Pasqua is a fellowship of National Council for Scientific and Technological Development (CNPQ).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Western diet increases HMGB1 levels and activates cardiac and hepatic Toll‐like receptor 4 (TLR4) signaling in male mice
Authors: Soultan Kimberly Mae , Carrillo‐Sepulveda Maria Alicia
Published: June 2021

Background

Overconsumption of a Western diet (WD), characterized by a high amount of fats and carbohydrates, is a major contributor to the epidemic of non‐alcoholic liver and cardiovascular disease in the United States. Our laboratory has recently demonstrated that WD causes endothelial dysfunction via vascular TLR4 signaling activation, a key component of innate immunity linked to inflammation. Based on these findings, we hypothesized that WD triggers inflammatory responses in the livers and hearts of male mice via activation of TLR4 signaling. We also investigated whether WD elevates high‐mobility group box 1 protein (HMGB1) levels, a pro‐inflammatory factor known to be an endogenous ligand for TLR4.

Methods

Adult male C57BL/J mice were randomized in two experimental groups: Control Group received a regular chow diet and WD group received WD (40% fat, 43% carbohydrates, and 17% protein) for 28 weeks. Prior to terminal experiments, body weights were measured weekly. After euthanasia with isoflurane (via nasal 5% in 100% O2), livers, hearts, and adipose tissue were removed and processed for further molecular assays. Blood samples were also obtained for biochemical analysis.

Results

As expected, WD group exhibited increased body weight (31.1 ± 3.2 vs. 25.2 ± 1.4g, p<0.01), adipocyte size (7724.2 ± 1745.8 vs. 2727.3 ± 927.1 μm2, p<0.05) and elevated serum free fatty acids (0.20 ± 0.02 vs. 0.14 ± 0.024 mM, p<0.05) compared to controls. Next, to test the hypothesis that WD activates TLR4 signaling in hepatic and cardiac tissue, Western blot analysis was performed. Hepatic expression of TLR4 (2.8 fold increase vs. controls, p<0.001) and its downstream protein TNF receptor‐associated factor 6 (TRAF6) (1.8 fold increase vs. controls, p<0.01) were markedly elevated in the WD group. Similarly, cardiac expression of TLR4 (2.2 fold increase vs. controls, p<0.01) and TRAF6 (2.8 fold increase vs. controls, p<0.001) were increased in the WD group. Strikingly, we found that serum HMGB1 expression was significantly increased in WD group in comparison to the controls (70% elevation vs control, p<0.05).

Conclusion

Our results suggest that upregulation of HMGB1 may contribute to TLR4/TRAF6 signaling activation in hepatic and cardiac tissue of western diet fed mice. To expand this investigation, we will utilize pharmacological and molecular approaches to target HMGB1 and confirm its role in WD‐induced inflammation.

Support or Funding Information

In‐house Grant NYIT

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dexamethasone regulates Hox and paraHox gene expression in mouse liver
Authors: Ji Yue , Cheng Xingguo
Published: June 2021

The homeobox (Hox) genes encode homeodomain proteins, which control the development of body forms of many organisms including plants, Drosophila, mouse, and human. Dexamethasone (DEX), a synthetic glucocorticoid with potent anti‐inflammatory effects, can activate pregnane X receptor (PXR) and glucocorticoid receptor (GR) nuclear receptors in a dose‐dependent manner. At low doses such as 2 mg/kg, DEX activates only GR in mouse liver. However, at higher doses such as 50 mg/kg, DEX activates both GR and PXR nuclear receptors. Few studies have reported that DEX can regulate the expression of Hox genes. The purpose of this study was to determine the regulation of Hox (including Hox a‐d subfamily members) and paraHox genes by DEX in mouse liver, as well as the underlying mechanism. Adult male C57BL/6 mice were treated with DEX (2 and 50 mg/kg) once daily for 7 days (n=4–6/treatment). Our results showed that: 1) 2 mg/kg of DEX did not alter mRNA expression of any Hox a–c subfamily members. 2) 50 mg/kg of DEX induced mRNA expression of many Hox a (including Hox a2, a3, a9, a10, a11, and a13), Hox b (including Hox b2, b4, b5, b6, b7, and b9), and Hox c (including Hox c4, c5, c9, c10, c11, and c13) subfamily genes. 3) 2 mg/kg of DEX induced mRNA expression of Hox d9, d11, and d12; whereas 50 mg/kg of DEX induced mRNA expression of Hox d1, d3, d4, d8, d9, d10, d11, and d12. And 4) DEX at the dose of 50 mg/kg but not 2 mg/kg, induced mRNA expression of several other Hox and paraHox genes, including Hex, Pbx1, Gsh1, and Cdx. In conclusion, DEX at the dose of 50 mg/kg but not 2 mg/kg up‐regulated mRNA expression of many Hox and paraHox genes in mouse liver, indicating that signaling of PXR but not GR is involved in Hox and paraHox gene expression.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.