bluuebunny/crossref_metadata_2025 huggingface dataset:
TMED9 is a poor prognosis marker in hepatocellular carcinoma patients that modulates oncogene trafficking and promote tumorigenesis
Authors: Yang Yi‐Chieh , Lai Tsung‐Ching , Hsiao Michael
Published: June 2021

Purpose

Hepatocellular carcinoma (HCC) is the third leading cause of cancer‐related deaths in the world. Due to difficulty of early diagnosis and the treatment in HCC, the prognosis for patients remains poor in the past decade. Therefore, our aim in this study is to establish the screening model to discover novel diagnostic and prognostic markers in HCC patients.

Design

Systemic analysis of global gene expressions by using Mass Spectrometry is an attractive approach to identify novel biomarkers for the development of therapeutics and diagnostics. In this study, the fresh protein lysates from paired N/T HCC tissues were analyzed by SDS‐PAGE protein MS/MS and tissue MALDI imaging analysis to identify the putative biomarkers. On the other hand, the frozen sections were applied to MALDI imaging MS system and analyzed by Fleximage and ClinPro software. Selected biomarker signatures were evaluated by the available online databases and immunohistochemistry staining was used to stain N/T paired clinical hepatoma tissue array (n=120) for prognostic value analysis. Immunoprecipitation‐MASS was further used to find the identified marker TMED9 binding proteins.

Results

According to the protein ID analysis, 27 proteins were revealed with more than 1.3 fold of T/N ratio. We further validated these genes by TCGA and GEO databases, which recruited the progression data and the paired N/T sample analysis in their cohort. Among these proteins, we found the importance of the TMED9 in HCC progression. The higher TMED9 mRNA and protein levels were significantly observed in the HCC patients. In addition, the patients who had higher TMED9 accompanied with poor prognosis in our Taiwan cohort. Mechanically, the dyregulation of TMED9 in HCC cells altered the trafficking of targeting proteins from ER to Golgi and may also changes their folding, maturation, and post‐translational modifications to modulate their protein functions or stability. The alteration of TMED9 expression changes the metabolism of functional proteins and promotes the tumorigenecity of HCC.

Conclusion

Our work not only identified the new protein biomarkers in predicting HCC prognosis, but also showed dyregulation of protein trafficking system could promote HCC progression. These new findings may provide new targets for therapeutic intervention in treating patients with HCC.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ghrelin Signaling Axis Associates with Poor Prognosis and Promotes Renal Cell Carcinoma Invasion
Authors: Lin Tsung‐Chieh , Hsiao Michael
Published: June 2021

Increasing studies point out a peptide hormone, ghrelin, playing critical roles in regulating cancer progression. Ghrelin is originally discovered to be expressed at stomach, and functions as an endogenous ligand of growth hormone secretagogue receptor (GHS‐R), which induces growth hormone (GH) release and appetite. Our group previously studied its prognostic significance in cancer patients, and the pro‐metastatic activity in animal model of clear cell subtype of renal cell carcinoma (ccRCC). However, ghrelin's potential function in regulating cell invasion remains obscure. In this study, we performed Ingenuity Pathway Analysis (IPA)‐based analysis of differentially expressed genes in Cancer Cell Line Encyclopedia (CCLE) dataset, which indicates the potential association of Aurora A with ghrelin in ccRCC metastasis. Furthermore, a positive correlation of ghrelin and Aurora A expression level has been confirmed in the dataset including 15 ccRCC cell line and in our ccRCC cell line model. ccRCC patients identified with high ghrelin and Aurora A status are clinically correlated with poor outcome. We further characterized that ghrelin could increase Aurora A level at both protein and RNA level, and the ghrelin‐induced ccRCC in vitro invasion and in vivo metastasis is dependent on Aurora A. To dissect the molecular mechanism, MMP1, 2, 7, 9, 10 and 11 expressions are increased upon ghrelin treatment. Among those matrix metalloproteinases (MMP), MMP10 level is increased and required for the ghrelin‐Aurora axis‐mediated ccRCC invasion. The results discovered in this study indicates a novel signaling mechanism in ccRCC metastasis.

Support or Funding Information

Ministry of Science and Technology (107‐2314‐B‐182A‐158 ‐MY3), Taiwan to Dr. Tsung‐Chieh Lin.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lactococcus lactis sp. cremoris elicits protection against metabolic changes induced in females by a Western style diet
Authors: Naudin Crystal , Saeedi Bejan , Owens Joshua , Darby Trevor , Jones Rheinallt
Published: June 2021

A high sugar and high‐fat diet Western‐style diet can result in significant dyslipidemia, often leading to non‐alcoholic fatty liver disease (NAFLD), with females particularly affected by this diet irrespective of total caloric intake. Dietary supplementation with beneficial bacteria has been advocated as therapeutic intervention to modulate liver adiposity resulting from a Western‐style diet. Here, we assess the activity of beneficial bacteria on modulating the impact of a Western‐style diet in female mice. Of those tested, we show that a previously uncharacterized beneficial bacterium, namely Lactococcus lactis sp. cremoris significantly protected against Western style diet‐induced hepatic steatosis, elevated cholesterol levels, glucose intolerance, increased body mass index (BMI), and adiposity. Due to these effects, we propose the use of L. lactis sp. cremoris as a therapeutic modality to promote metabolic health in individuals suffering adverse health events resulting from a Western style diet.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Contribution of Fractalkine to Incorrect Angiogenesis in Preeclamptic Placentas
Authors: Szukiewicz Dariusz , Szewczyk Grzegorz
Published: June 2021

Disregarding the multiple research in the field of preeclampsia, the exact pathology remains unclear. The shallow invasion of extravillous trophoblast (EVT) in spiral arteries leads to the development of pregnancy‐induced hypertension, nevertheless the etiological factor is unknown. Disturbed differentiation of EVT and incorrect placental angiogenesis seem to be one of the most important starting points for pathogenetic and etiological concepts in preeclampsia. As fractalkine (chemokine CX3CL1) by the activation of its only receptor CX3CR1 stimulates angiogenesis and integrin‐dependent cell migration, the authors ask the question about the possible role of CX3CL1 in placental angiogenesis in preeclampsia. A total number of 58 pregnant patients were included into the study, preeclampsia was diagnosed in 29 patients, and 29 patients were healthy, normotensive term pregnancies. In all women between 30 and 32 week of pregnancy CX3CL1 level was assessed in plasma using ELISA and the expression of CX3CR1 and angiogenesis score (V/EVTI: vascular/extravascular tissular index) were assessed in placental tissues obtained after delivery. Quantitative immunohistochemistry was applied for assessment of both CX3CR1 expression and V/EVTI in placental sections. For this second purpose, morphometric measurements of the placental microvasculature were performed after immunohistochemistry of endothelial cells with CD‐34 antibody. The average duration of pregnancy in preeclamptic group was shorter by 5,3 weeks than in normotensive pregnancies. CXC3CL1 plasma concentration was significantly lower in preeclamptic patients (mean 279 pg/ml ± 243 vs mean 846 pg/ml ± 418), whereas CX3CR1 expression was significantly (p < 0.05) higher in preeclamptic group. A negative correlation between CX3CL1 level and CX3CR1 expression was observed. Moreover, the V/EVTI value was lowered in preeclampsia (p < 0.05). In conclusion, disturbed placental angiogenesis in preeclampsia may be associated with low plasma fractalkine level.

Support or Funding Information

WUM grant: 2M2‐W1‐18

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Detailed Explanation of the Distribution of Atherosclerosis in the Pelvic Cavity
Authors: Khan Sameer Ahmad , Savinova Olga , Beatty Brian Lee
Published: June 2021

Atherosclerosis is an important indicator for future pathological conditions such as stroke, peripheral artery disease, and coronary ischemic events. Though it is known that atherosclerosis does not present in the systemic circulation evenly, detailed studies of many vascular regions are lacking. In this study, we analyze the atherosclerotic distribution present in the iliac arteries, an area of interest because of the known prevalence of ischemic events in lower extremities and pelvic area ‐ claudication, overactive bladder, and bladder pain syndrome. While hematoxylin and eosin (H&E) stain enables pathophysiological staging of atherosclerotic lesions, it does not illustrate the distribution of calcium deposition in a blood vessel due to low sensitivity and a lack of specificity for calcium. In this study we used alizarin red stain ‐ a histological stain specific for calcium – to increase the detection of calcium deposition in vascular tissues. We used ImageJ analysis to determine percent of alizarin red – positive area (as a measure of calcification) in 6 vessels from 25 cadavers. These vessels were common iliac, external iliac, posterior division on internal iliac, anterior division of internal iliac, superior gluteal, and inferior gluteal arteries. We observed a higher percentage of calcification in the tunica intima rather than the tunica media. We also found a correlation between the percentage of calcification in tunica intima in the common iliac and anterior divisions, as well as the common iliac and inferior gluteal arteries. In tunica media, we found correlation between calcification in the common iliac artery and all other vessels examined except the external iliac. Correlations were also found between medial arterial calcification in external iliac artery and all other vessels (except the common iliac). We found that there are three pairs of vessels, external iliac and posterior division, external iliac and superior gluteal, and external iliac and inferior gluteal arteries with significant medial layer calcification correlations within each subject. Each of these pairs of vessels are significantly far away from each other in the branching pattern. Though speculative, this data could be used in future pathophysiological studies of atherosclerosis both in the pelvic cavity and systemically.

Support or Funding Information

NYIT College of Osteopathic Medicine Summer Research Program

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Construction of a Common Medication Formulary and Prototype Drug Information for Use in Undergraduate and Graduate Nursing Program Curricula
Authors: Meslin Coralie , Miller Katherine , Veidenheimer Paige , Murphy Patrick J. M.
Published: June 2021

An essential element in pharmacology course development is the selection of exemplar medications, which are introduced to students as prototype drugs and intended to be representative of the mechanisms, uses, and effects of entire pharmacological categories seen in clinical practice. The purpose of this project was to construct a clinically relevant and pedagogically sound prototype list and most pertinent drug information that could be incorporated across undergraduate and graduate nursing program curricula. Studies have found numerous barriers to effective pharmacologic learning in nursing undergraduate and graduate students, including content saturation and a reliance of memorization‐heavy teaching techniques. While physician‐directed pharmacology education has benefited from consensus pharmacology knowledge objectives and drug lists compiled by the Association of Medical School Pharmacology Chairs, no such counterpart exists for nursing. The concept‐based model of pharmacology education favors using a prototype drug to teach general information for the drug class, and has been proven to effectively respond to proven barriers for pharmacologic learning for nursing students. We developed a formulary to be used by registered nurse (RN), advanced registered nurse practitioner (ARNP), and physician assistant (PA) students utilizing a concept‐based model of pharmacology education. The curated material, which emphasizes medications relevant to family practice and primary care, presents the most applicable nursing interventions, monitoring parameters and side effects for the most commonly used prototype drugs. We collated a list that represents the most commonly used drugs, most frequent and most concerning side effects, as well as pharmacologic information germane to RN, ARNP, and PA licensure examinations. By highlighting these key features of medications, we created a resource that can be used by nursing educators to better serve and educate their students.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Morphine Induces Differential Activation and Cellular Localization of Protein Kinases in the Mouse Periaqueductal Gray
Authors: Ram Akila , Edwards Taylor , Bobeck Erin Nicole
Published: June 2021

Morphine is a commonly used potent opioid analgesic with very high propensity for tolerance development. The analgesic and tolerance properties of opioids are partially regulated by the midbrain ventrolateral periaqueductal gray (vlPAG). Here, we investigate the intracellular kinase activation and localization patterns in the mouse vlPAG following acute and chronic morphine treatment. Wild‐type mice were treated with morphine (10 mg/kg, i.p.) or saline in a chronic or acute administration paradigm. We performed fluorescence immunohistochemistry to map the localization and activation of extracellular signal‐regulated kinase 1/2 (ERK 1/2), protein kinase‐C (PKC), and protein kinase‐A (PKA). We observed significantly greater activated ERK 1/2 in the vlPAG chronic morphine‐treated animals. We also visualized the co‐localization of ERK 1/2 with the endosomal marker Eea1. We note that phosphorylated PKC tends to localize to the plasma membrane in the vlPAG following chronic morphine treatment and interacts with the μ‐opioid receptor. Further, we observed that although PKA activation is increased following chronic morphine treatment, there is a significant reduction in nuclear translocation of this kinase. Additionally, we examined the cell‐type specificity of kinase activation by co‐localizing these kinases to GAD67 and VGluT neurons that project from the PAG downstream to the Rostral Ventromedial Medulla. Taken together, this study demonstrates a differential activation and localization of ERK 1/2, PKC and PKA, and opens avenues to explore the role of chronic morphine treatment on G‐protein signaling and kinase nuclear transport.

Support or Funding Information

This research is supported by the Department of Biology at Utah State 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.
In vivo Dependence Liability Assessment of Novel Fentanyl Analogues: Science to Guide drug Policy
Authors: Urquhart Kyle , Fukuda Saki , Gogoi Juoti , Janowsky Aaron , Eshleman Amy , Hiranita Takato , Fantegrossi William E.
Published: June 2021

The emergence of fentanyl analogues as drugs of abuse raises concern regarding their dependence liability. Following prolonged opioid administration, physical dependence leads to increased use, which facilitates the development of tolerance and the dangers of overdose. In animal models, opioid dependence can be inferred by assessing signs of withdrawal, with vertical jumping being the most sensitive and easily quantified in the mouse. In this study, we aimed to characterize the dependence liability of fentanyl, morphine, and six fentanyl analogues by assessing antinociception and withdrawal signs elicited by each compound in the mouse. First, to determine antinociceptive effects, we used a warm‐water tail withdrawal procedure using both 50°C and 55°C water. Latencies until mice removed their tails from the water were recorded, and fully effective doses were determined using a 10 second cut‐off to prevent tissue damage. Dose effect‐curves were generated to determine the potency (ED50) of each compound for eliciting antinociception. All analogues were less potent than fentanyl but more potent than morphine. Additionally, all test compounds were fully effective in both 50°C and 55°C water. Next, to assess withdrawal, we treated animals with 3 injections of morphine, fentanyl or the various fentanyl analogues spaced 12 hours apart. Following the first two injections on day 1, we precipitated withdrawal on day 2, 3 hours after the final injection of agonist, using the nonselective opioid antagonist naloxone. Withdrawal sessions lasted 20 minutes and the number of vertical jumps were recorded for each session. Antagonist dose‐effect curves for withdrawal jumps were generated first using an agonist dose one‐log unit higher than the fully effective antinociceptive dose and precipitating withdrawal with 3, 10, and 30 mg/kg of naloxone. Next, we used 30 mg/kg of naloxone to precipitate withdrawal using increasing unit doses of each test compoewund since maximal jumping was elicited using 30 mg/kg of naloxone. Agonist dose‐effect curves were generated starting with the lowest fully effective antinociceptive dose and increasing in half‐log units. Our results indicate that all of these fentanyl derivatives are capable of eliciting dependence and offer valuable insight into their abuse potential.

Support or Funding Information

These studies supported in part by DEA/NCTR (224‐16‐0505R),) and the University of Arkansas for Medical ScienVA (D‐15‐OD‐002ces

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 Differences in Heroin Self‐Administration Behaviors and Heroin‐Induced Receptor Alterations in Rats
Authors: George Brianna , Jones Sara
Published: June 2021

Despite reports that women are more vulnerable than men to certain features of heroin addiction, including craving and relapse, there are limited numbers of preclinical studies evaluating these phenomena. In addition, the published sex‐comparison studies in preclinical models of heroin addiction have shown conflicting results. For example, Lynch and Carroll (1999) report that female rats acquire heroin self‐administration faster than males, while Stewart et al. 1996 report no differences in acquisition rates between male and female rats. Therefore, this study examined sex differences in several heroin self‐administration behaviors and putative neural correlates. Adult male and female Sprague‐Dawley rats were implanted with jugular catheters and were given access to heroin (FR1 schedule of reinforcement, 0.025 mg/kg/inf; maximum 20 infusions) for 6 hours per day. Acquisition criteria were met when rodents earned all 20 infusions for 2 consecutive days. After acquisition, all animals were placed on a long access (LgA) paradigm, which included unlimited access to heroin (FR1) for 6 hours/day for 10 consecutive days. After completion of LgA, animals were tested for motivation to self‐administer heroin using a progressive ratio (PR) schedule. Finally, we measured basal dopamine dynamics and the ability of kappa opioid receptor (KOR) and D2/D3 autoreceptor agonism to reduce dopamine release in the nucleus accumbens following exposure to heroin self‐administration using ex vivo fast scan cyclic voltammetry (FSCV) in brain slices. Female and male rats acquired heroin self‐administration at similar rates, but females self‐administered more heroin during LgA, had increased loading doses of heroin during the first 10 minutes of LgA sessions, and increased escalation of intake across the 10 LgA sessions compared to males. PR data revealed a rightward shift in the dose‐response curve of the females, indicative of increased motivation to obtain higher doses of heroin. Using FSCV, we found that all rats displayed greater KOR‐ and D3 autoreceptor‐mediated dopamine release inhibition, however, females showed higher levels of activity for both receptors. These results reveal marked sex differences in specific heroin self‐administration behaviors that may be driven by altered receptor functioning within the nucleus accumbens. Future studies elucidating receptor interactions related to sexually dimorphic heroin self‐administration behaviors in rats will be highly informative.

Support or Funding Information

T32 DA041349

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Applying label‐free quantitative proteomics to identify profile protein changes in hippocampus of rats after chronic cerebral hypoperfusion
Authors: Wang Yue‐Hua , Yang Ying‐Lin , Cheng Xiao , Lyu Yang , Du Guan‐Hua
Published: June 2021

Vascular dementia (VD) refers to a progressive decline in memory and cognitive function caused by chronic cerebral hypoperfusion. 2‐vessels occlusion (2‐VO) induced by bilateral common carotid artery occlusion has been widely used as a model of VD. In the present study, the global proteins profile of hippocampus in 2‐VO rats was studied using label‐free quantitative proteomics. The results showed that 2‐VO induced learning and memory deficits, motor ability dysfunction, and neuronal plasticity injury in rats. The proteomics results showed that there are involved in up‐regulation of 55 proteins and down‐regulation of 171 proteins in the hippocampus of 2‐VO group vs that of sham group. Gene ontology and signal pathway analysis showed that the regulated proteins are mainly involved in intracellular signaling cascade, metabolic process, oxidation reduction process, oxidative phosphorylation, etc. Furthermore, RT‐PCR results revealed that the mRNA expression of Mt‐nd1, Serpine2, Mrpl15 was significantly decreased and whereas Itapka, Ptpn1, Abracl, Gorasp2 were significantly increased in the hippocampus of 2‐VO rats compared with sham rats. WB result further demonstrated MT‐ND1 expression was consistent with the results of proteomics. The current findings provide new insights into the molecular mechanisms of chronic cerebral ischemia.

Support or Funding Information

National Key Research and Development Program of China (2018YFC0311005), National Natural Science Foundation of China (81473383), National Science and Technology Major Project of China (2018ZX09711001‐003‐019), CAMS Innovation Fund for Medical Sciences (2016‐I2M‐3‐007), and Innovation Fund for Graduate of Beijing Union Medical College (2017‐1007‐02; 2018‐1007‐04).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Claudin‐5 as a target for drug delivery to the brain by modulation blood‐brain barrier
Authors: Kondoh Masuo , Hashimoto Yosuke , Shirakura Keisuke , Okada Yoshiaki , Takeda Hiroyuki , Endo Kohki , Tamura Maki , Watari Akihiro , Sadamura Yoshifusa , Sawasaki Tatsuya , Tachibna Keisuke , Doi Takefumi , Yagi Kiyohito
Published: June 2021

The regulatory success rate for central nervous system (CNS) drugs is less than half that of non‐CNS drugs. A current bottleneck in the development of CNS drugs is the lack of drug delivery systems to the brain. The blood‐brain barrier (BBB) is a membrane barrier formed by brain capillary endothelial cells and protects the brain from harmful materials circulating in the blood. Most small and all large drugs do not readily cross the BBB. The intercellular space between endothelial cells of the BBB is sealed by complex protein‐based structures called tight junctions (TJs). Claudin‐5 (CLDN‐5), a tetra‐transmembrane protein, is a key component of the TJ seal that prevents the paracellular diffusion of drugs into the CNS. Here, to investigate whether CLDN‐5 can be a target for CNS delivery of drugs, we generated monoclonal antibodies (mAbs) specific to the extracellular domain of CLDN‐5 using DNA immunization. Treatment of MDCKII/CLDN‐5 cells with anti‐CLDN‐5 mAbs time‐ and dose‐dependently decreased the integrity of TJs. The integrity was restored by removal of the anti‐CLDN‐5 mAbs. In an in vitro model of the BBB, anti‐CLDN‐5 mAbs also attenuated the integrity of TJs and enhanced permeability of small (Mw 376 Da) and large (4 kDa) solutes. The anti‐CLDN‐5 mAbs did not decrease TJ integrity in human intestinal epithelium models. In conclusion, CLDN‐5 is a potential target for the development of drug delivery systems to the brain. The anti‐CLDN‐5 mAb is a potential lead compound for development of novel drug delivery systems targeting the CNS.

Support or Funding Information

This study was partly supported by Grant‐in‐Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (18K19400, 18H03190, 16K13044).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Honokiol Attenuates Amyloid‐beta Induced Neuroinflammatory Microglial Polarization in BV2 Microglial Cells
Authors: Jones Ellery , Govindarajulu Manoj , Ramesh Sindhu , Dhanasekaran Murali
Published: June 2021

Neuroinflammation plays a crucial role in the pathogenesis of Alzheimer's Disease (AD). Microglia are the macrophages of the central nervous system (CNS), which play a crucial role in immune surveillance and maintaining homeostasis. Chronic microglial activation is detrimental because they produce several pro‐inflammatory cytokines, which result in neuronal cell death (M1 phenotype). Additionally, several studies indicate that anti‐inflammatory mechanisms are down‐regulated in AD (M2 phenotype). Honokiol is a lipophilic polyphenol known to possess potent anti‐inflammatory properties. The goal of this study was to determine the effect of honokiol on activated microglia. The microglial cell line BV2 was pretreated with Honokiol, followed by treatment with amyloid beta, for 24 hours. Pro‐inflammatory activation was assessed by measuring nitric oxide, TNF‐α and IL‐6 levels. Biochemical assays, including western blot, immunocytochemistry and PCR, were performed to mechanistically determine the effect of honokiol on microglial activation. Honokiol significantly reduced NO, ROS, TNF‐α and IL‐6 levels. Honokiol also significantly reduced iNOS expresssion, down‐regulated markers of M1 phenotype and increased M2 markers. The promotional effects of Honokiol on M2 polarization were attenuated by an AMP‐activated protein kinase (AMPK) inhibitor, indicating that AMPK activation participates in Honokiol's effects. Furthermore, Honokiol‐mediated M2 gene expression was prevented by an inhibitor of calmodulin‐dependent protein kinase kinase β (CaMKKβ), an upstream kinase of AMPK. Our in vitro studies indicate that Honokiol could be acting as an AMPK activator, thereby enhancing M2 polarization and suppressing microglial activation. Further studies in animal models need to be conducted to validate Honokiol as a novel and safe therapeutic compound for the treatment of neuroinflammation associated with AD.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Monitoring Signalling and Trafficking of Neurotensin Type 1 Receptor in Animal Model using Fluorescent‐based Methods
Authors: Couvineau Pierre , Ehrlich Aliza , Semache Meriem , Kieffer Brigitte , Bouvier Michel
Published: June 2021

Neurotensin Type 1 Receptor (NTSR1) is a G Protein‐Coupled Receptor (GPCR) with proposed roles in cancer, neuropsychiatric and neurodegenerative diseases, eating disorders, pain, as well as inflammation. It therefore represents a potential therapeutic target for the treatment of these diseases. However, relatively little is known about the repertoire of signalling effectors that can be engaged by NTSR1 and about the functional selectivity of drug candidates targeting this receptor in native tissue.

In a first step toward generating an animal model allowing to monitor the activation and regulation of NTSR1 in native tissues, we developed a DNA construct expressing NTSR1 tagged to vYFP at its C‐terminus.

We investigated the signalling responses of NTSR1‐vYFP promoted by eight agonists and two antagonists, and compared them to those obtained for the unmodified NTSR1. For this purpose, we used a suite of BRET‐based sensors expressed in HEK‐293 cells. We showed that NTSR1‐vYFP has the same signalling profile as the endogenous NTSR1 as it activates both β‐Arrestin 1 and 2 and all G proteins except G12 and Gi3. Moreover, we confirmed that ML314 is a biased agonist toward β‐Arrestin pathways and we showed that PD149163 does not activate Gs protein.

Because the vYFP fusion does not seem to modify the signalling profile of the NTSR1, we have embarked on developing a knock‐in mouse model expressing NTSR1‐vYFP instead of the native receptor which will be a useful tool to study receptor signaling and trafficking under physiological conditions.

Support or Funding Information

The project is funded by the Consortium de recherche biopharmaceutique (CQDM). Pierre Couvineau is funded by the INSERM‐FRQS Postdoctoral Exchange Program

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression of different adenylyl cyclase isoforms impacts ligand bias downstream of the kappa opioid receptor
Authors: Brust Tarsis , Scarry Sarah , Aubé Jeffrey , Bohn Laura
Published: June 2021

Functionally selective (or biased) ligands have been widely pursued in recent years as a way to target G protein‐coupled receptors (GPCRs) in a more selective manner. The promise of biased signaling is that by specifically activating only a subset of a receptor's responses, therapeutic effects can be achieved and adverse effects prevented. Molecular studies of biased ligands are commonly carried out in cell lines, and many of them use cyclic AMP accumulation as a readout of G protein activation. The production of cyclic AMP is catalyzed by adenylyl cyclases (ACs). In humans there are 10 different isoforms of ACs, which diverge in their expression patterns and regulatory properties. In the present study, we examined if the expression of different AC isoforms in a cell line could result in different bias profiles for ligands of the kappa opioid receptor (KOR). We employed one biased and two reference ligands and showed that there were significant changes in potency as well as in bias factors depending on the AC isoform that was expressed in our cellular system. Our results support the idea that selection of an appropriate cellular model is an invaluable step in studies of functional selectivity. Moreover, using cells that match the AC expression pattern of the tissue where the compound is predicted to act can lead to more relevant results when cyclic AMP is used as a readout of G protein activation.

Support or Funding Information

This work was supported by National Institute on Drug Abuse grant R01DA031927.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Aldosterone binding to G Protein Estrogen Receptor‐GPER
Authors: Ding Qingming , Chorazyczewski Jozef , Gros Robert , Feldman Ross
Published: June 2021

MR‐independent effects of aldosterone acting through GPER have been appreciated since 2011. However, whether this related to direct aldosterone‐GPER binding has been disputed. Two previous radioligand studies failed to demonstrate direct aldosterone binding to a putative GPER. However, in neither study was it demonstrated that the radioligand used, bound to a physiological receptor. Further, both utilized 3H estradiol‐ whose low potency for GPER binding would not be expected to allow GPER receptor identification using vacuum filtration methods related to its high off‐rates. Therefore to determine if aldosterone did bind to GPER, we studied its displacement of [3H] 2‐methoxyestradiol binding ([3H]2ME ‐ a higher affinity GPER‐selective agonist) in SF9 cells with heterologous GPER expression mediated by baculovirus.

In this model system, following GPER gene transfer, [3H]2ME demonstrated saturable and reversible binding to a high affinity population of receptors with a Kd = 3.4±0.4 nM and Bmax = 645±49 fmol/mg. In the absence of baculovirus GPER gene transduction, no high affinity [3H]2ME binding was demonstrable in SF9 cells. [3H]2ME binding was guanine‐nucleotide sensitive with a 43±8% reduction in high affinity binding in the presence of GppNHp (100μM). High affinity [3H]2ME binding was displaceable by 2ME [with an IC50 comparable to the determined Kd (1.3±0.4 nM)], the GPER agonist G1, the GPER antagonist G15, estradiol (E2) and aldosterone (Aldo) but not by hydrocortisone (HC) with an order of potency of (2ME>Aldo>E2=G1>G15>>HC).

In summary, we demonstrate high affinity [3H]2ME binding to GPER with an order of potency for aldosterone vs. estradiol (vs. hydrocortisone) that parallels the order of potency of their functional effects in previously studied models. These data support the hypothesis that aldosterone mediates its GPER‐dependent effects by direct binding to the receptor.

Support or Funding Information

Heart and Stroke Foundation of Canada

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
15,16‐Dihydrotanshinone I, a natural product, protects ischemic stroke by inhibiting ferroptosis
Authors: Li Shaojing , Wu Chuanhong , Chen Xiuping
Published: June 2021

Background/Aims

Ischemic stroke is a leading cause of death and disability worldwide. Early intervention of cell death in the penumbra can greatly reduce the infarct volume, thereby, alleviating the ischemic progress. Ferroptosis is a form of regulated cell death that is dependent on iron and reasctive oxygen species and is characterized by lipid peroxidation. Mitochondrial ROS are of importance in ferroptosis. Recent studies showed that it also implicated in the pathological cell death associated with stroke. Here, we studied the effect of 15,16‐Dihydrotanshinone I (DHT), a lipophilic tanshinone extracted from the root of Salvia miltiorrhiza Bunge (Danshen), on ischemic stroke and its underlying mechanism.

Methods

Permanent middle cerebral artery occlusion (pMCAO)‐induced focal cerebral ischemia rats and tert‐butyl hydroperoxide (t‐BHP) injured PC12 cells were used to interpret the neuroprotective effect, especially the anti‐ferroptosis effect of DHT in vivo and in vitro.

Results

The results showed that DHT protected t‐BHP injured PC12 cells and reversed ferroptosis induced by t‐BHP in vitro. It also protected rats subjected to pMCAO from ferroptosis in vivo. Furthermore, DHT ameliorated neurological score, infarct volume, cerebral blood flow, cerebral edema and microstructure of white‐grey matter in pMCAO rat in vivo.

Conclusion

The data in this study demonstrated that DHT might have therapeutic potential for ischemic stroke and play its protective role against ferroptosis. This study reported that ferroptosis in the ischemic stroke represents a potential therapeutic target for the first time, and it might provide new insight into the prevention of ischemic stroke.

Support or Funding Information

the Science and Technology Development Fund of Macau Special Administrative Region (FDCT) (No. 078/2016/A2) , the Research Fund of University of Macau (MYRG2016‐00043‐ICMS‐QRCM), the National Natural Science Foundation of China (No. 81274133), the Major Scientific and Technological Special Project for “Significant New Drugs Creation” (No. 2018ZX09201008‐001, 2012ZX09103‐201‐055, ), the Fundamental Research Funds for the Central public welfare research institutes of China (ZZ2014060,ZXKT17040)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Antipsychotic Drug, Risperidone, Distributes to Bone Marrow: What is the Impact on Immune Function?
Authors: Houseknecht Karen L. , May Meghan , Beauchemin Megan , Barlow Deborah , Geguchadze Ramaz
Published: June 2021

Antipsychotic medications are associated with serious side effects including obesity, dyslipidemia, diabetes, increased risk of fractures and bone loss. Additionally, it has been widely reported that schizophrenic patients are more susceptible to infections; however the role of antipsychotic medications (if any) in determining susceptibility to infectious disease is not understood. We have previously reported that risperidone (RIS) distributes to bone marrow of female mice following a single oral dose (Motyl et al., 2017, Bone 103:168–176).

Hypothesis

The bone marrow compartment may be a key target of risperidone action, resulting in altered immune function.

Methods

To test this hypothesis pre‐clinically, we treated male and female C57BL/6 mice with vehicle (VEH) or a low, clinically relevant dose of RIS (0.75 mg/kg, PO) for 5 days or 4 weeks. At the end of each treatment period, mice were sacrificed and plasma, bone marrow, and other tissues were collected for analysis. All animal study protocols were approved by the University of New England IACUC committee.

Results

Concentrations of drug and metabolites in plasma and bone marrow were determined by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) analysis. RIS and the active metabolite 9‐OH RIS (PAL) distributed to marrow of male mice following 5 days of dosing, and concentrations were higher in marrow than in plasma at 1 and 3 hours post‐dose (10–15 fold higher in marrow vs. plasma; P<0.05). Plasma concentrations of 40 cytokines or chemokines were measured in plasma from mice treated for 4 weeks with RIS or VEH using the Mouse Cytokine Array Panel (Proteome Profiler*TM Array, R&D Systems, Minneapolis, MN). Eight key immune mediators (IL‐17, INFγ, IL‐7, TIMP1, BCA‐1, IL‐13, IL‐1α, and I‐309) were significantly (P <0.5) downregulated in RIS‐treated mice. Hearts from VEH and RIS mice treated for 4 weeks were analyzed by RNASeq analysis and expression of 31 genes from immune/inflammatory pathways were significantly (2‐fold; P<0.05) altered (8 upregulated; 23 downregulated) in a manner consistent with global immunosuppression.

Conclusions

The highly prescribed antipsychotic, risperidone, distributes to bone marrow in mice and is associated with global immunosuppression as evidenced by reduced tissue (heart) and plasma concentrations of multiple cytokines and immune modulators. These data are consistent with increased susceptibility to infection, observed clinically. Future studies will explore the effect of RIS to modulate response to an immune challenge (in vivo).

Support or Funding Information

Funding source: NIH grant DK095413 to KLH

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sodium propionate protects from inflammation and ROS stress
Authors: Lanza Marika , Casili Giovanna , Filippone Alessia , Campolo Michela , Paterniti Irene , Esposito Emanuela
Published: June 2021

The major end‐products of dietary fibre fermentation by the gut microbiota are the short‐chain fatty acids (SCFAs) acetate, propionate and butyrate, which have been shown to modulate host metabolism via effects on metabolic pathways and receptor‐mediated mechanisms at different tissue sites. The beneficial effects of SCFAs on various aspects of gut physiology, barrier function, and metabolism have been well documented. Furthermore, SCFAs can promote intestinal homeostasis and suppress intestinal inflammation. Recently, several reports have been published describing inhibitory effects of SCFA on NF‐κB, one of the key transcription factors regulating genes implicated in innate immunity, cell cycle control, and apoptosis. However, most previous studies mainly focused on butyrate, and few studies have devoted their efforts to other SCFAs such as propionate, although it is abundant as butyrate in the gut and blood. Thus, the purpose of this study is to investigate protective mechanisms of sodium propionate (SP) in inflammatory and oxidative stress responses. The first step was to evaluate the anti‐inflammatory and ant‐oxidant mechanism of SP, by an in vitro model on murine macrophage cell line, J774‐A1, by two different approaches: stimulation with lipopolysaccharide (LPS) from E. coli and H2O2, both followed by the treatment with SP at different concentrations (0.1–1 and 10 mM ). Moreover, to evaluate the effect on acute inflammation, an in vivo model of Carrageenan (CAR)‐induced rat paw inflammation was performed, where rats were orally treated with different doses of SP (10 mg/kg, 30 mg/kg and 100 mg/kg). Our in vitro results showed that SP (0.1‐1‐10mM) significantly decreased in concentration‐dependent‐manner the expression of pro‐inflammatory mediators, such as cyclooxygenase‐2 (COX‐2) and inducible nitric oxide synthase (iNOS) following LPS stimulation. Moreover, it has been demonstrated that SP (0.1‐1‐10 mM) was able to enhance anti‐oxidant enzyme production such as manganese‐dependent superoxide dismutase (MnSOD) and heme oxygenase‐1 (HO‐1) following H2O2 stimulation. In addition, in in vivo model, SP(30 mg/kg and 100 mg/kg) markedly reduced paw inflammation, thermal hyperalgesia and tissue damage induced by CAR injection. Our results clearly demonstrated the anti‐inflammatory and anti‐oxidant properties of SP, therefore, we propose that SP may be an effective strategy for the treatment of inflammatory diseases.

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.
Matrix Metalloproteinase‐9 Release from Porphyromonas gingivalis Lipopolysaccharide (LPS)‐treated Rat Brain Microglia In Vitro
Authors: Memedovski Zylfi , Czerwonka Evan , Liao Shiting , Han Jin , Mayer Joshua , Hall Mary L , Mayer Alejandro M
Published: June 2021

Background

The putative association between periodontal disease (PD), a chronic inflammatory disease and neuroinflammation, a condition which involves brain microglia, remains under investigation. We have reported that Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS), a bacterium implicated in periodontitis, activates both classical (M1‐type) and alternative (M2‐type) activation of neonatal rat microglia (BMG) with concomitant release of superoxide anion, thromboxane B2, cytokines, and chemokines (Faseb Journal 32.1 Supplement: 702.2, 2018). We hypothesized that matrix metalloproteinase 9 (MMP‐9) release by Pg‐treated microglia might be detectable by both ELISA and visualized by confocal fluorescence imaging.

Methods

P. gingivalis LPS (Pg LPS) 105 ng/mL from InvivoGen (San Diego, CA) and Escherichia coli LPS (Ec LPS) 026:B6 1 ng/mL from Difco Lab, Detroit, MI (positive control) were used to treat rat BMG for 18 hours at 35.9°C in vitro. MMP‐9 release was assessed with a rat MMP‐9 ELISA kit (R&D Systems). Confocal fluorescence imaging with a Nikon A1R laser confocal microscope was used to identify BMGs using a primary mouse anti‐rat CD11b/c antibody (Ab) (AbD SeroTec, Raleigh, NC), and secondary donkey anti‐mouse Ab (Thermo Fischer, Waltman, MA). MMP‐9 was visualized with a primary rabbit anti‐rat MMP‐9 polyclonal Ab, and secondary goat anti‐rabbit Ab, both from Abcam, Cambridge, MA.

Results

MMP‐9 presence in tissue culture supernates was detectable by ELISA in both Ec LPS and Pg LPS‐treated BMG. In contrast, MMP‐9 was observed by confocal fluorescence imaging only within Ec LPS‐treated BMG but not in Pg LPS‐treated BMG.

Conclusions

Both ELISA and confocal fluorescence imaging provide additional support for our working hypothesis, because Pg LPS stimulated BMGs to release MMP‐9. Thus, our current observations extend our studies on the effect of Pg LPS on rat brain microglia activation and contribute to further characterize the putative role of Pg LPS in both periodontitis, neuroinflammation and neurodegeneration.

Support or Funding Information

Support by College of Dental Medicine, College of Graduate Studies, and Chicago College of Osteopathic Medicine, Midwestern University are gratefully acknowledged.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interaction of Local Anesthetics with Hepatocellular Organic Anion Transporting Polypeptides
Authors: Stieger Bruno , Antener Enrica , Häusler Stephanie , Kullak‐Ublick Gerd A.
Published: June 2021

Organic anion transporting polypeptides (OATPs) have a very broad substrate specificity and are key transporters involved in drug disposition. Some OATPs display a broad tissue distribution. The substrates of OATPs are often anions, but also include neutral and cationic compounds and can be poorly water soluble. Local anesthetics can be divided chemically into amino esters and amino amides. Local anesthetics may undergo hepatic elimination and therefore require transport systems for hepatic uptake prior to metabolism. The aim of this project therefore is to test for a possible interaction of local anesthetics with the hepatocellular OATPs: OATP1B1, OATP1B3 and OATP2B1. Cells stably expressing hepatocellular OATPs (DMD 35: 1400, 2007; EJP 584: 57, 2008) were used for transport experiments. Inhibition of local anesthetics on the transport of standard OATP substrates was investigated as previously described (DMD 35: 1400, 2007; EJP 584: 57, 2008). So far, the two prototypic local anesthetics lidocaine (amine ester) and procainamide (amine amide) were tested using this approach. We found no interaction of these two local anesthetics on transport mediated by either of the three OATPs tested. We conclude that testing additional local anesthetics is necessary for testing our hypothesis.

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