bluuebunny/crossref_metadata_2025 huggingface dataset:
Exposure to Electronic Cigarette Vapor Causes Systemic Accumulation of Biomolecular Free Radicals
Authors: Clemons Garrett , Aboaziza Eiman , Burrage Emily , O'Reilly Juliana , Chantler Paul , Kelley Eric , Olfert Mark
Published: June 2021

Little is known regarding the impact of electronic cigarette (e‐cig) usage in the human body. However, emerging literature suggests that vaping results in endothelial cell mediated vascular impairments, altered thrombosis, and depressed immune function (similar to that observed with smoking). Since e‐cigs are marketed as a ‘safer’ alternative to traditional cigarettes, it is important to characterize mechanisms through which e‐cigs may induce cellular insult. It is known that traditional cigarette use results in systemic accumulation of biomolecular free radicals (BFR), thus we sought to determine whether exposure to e‐cig vapor will result in significant systemic increases in BFR. We hypothesize that vaping will increase the relative amount of BFR accumulation in vital tissues in a time‐dependent manner.

To identify BFR in‐vivo, C57BL/7 mice were given IP 1.5 g/kg DMPO (5,5‐dimethyl‐l‐pyrroline‐l‐oxide) spin‐trap in 2 doses over 1 hour, and acutely exposed to french vanilla‐flavored e‐cig vapor (18 mg nicotine) for either 1 hour (n=3) or 4 hours (n=3). Sham mice were exposed to ambient air (n=3). Immediately following exposure, the mice were anesthetized, perfused with PBS via intra‐ventricular catheterization, organs/tissues were harvested and fixed in paraformaldehyde/sucrose, flash‐frozen in liquid nitrogen and then fixed in TFM. Imaging from fluorescent immunohistochemical labeling revealed significant increase BFR accumulation in the liver (223%, p<0.05) and to lesser extent in the heart (133%, p<0.05) in 1‐hour exposed animals. Similar trends for increased BFR (albeit not statistically significant) were found in the lung (236%) and brain (124%) in 4‐hour exposed group compared to air exposed controls.

In summary, we found significant increase in the presence of BFR in the liver and heart of mice acutely exposed to 1‐hour of e‐cig vapor compared to controls, with similar trends in other tissue beds (lung, brain) after 4‐hours vape exposure. This is a pilot study which needs additional samples for further evaluation, but suggests that vaping induces BFR accumulation in multiple organs which could lead to cellular damage and tissue dysfunction.

Support or Funding Information

Support: WVU Cancer Institute Philip R Dino Innovative Research Grant (IMO); Transition Grant Support; Office of Research and Graduate Education (IMO), WVU Health Sciences Center APS STRIDE Fellowship (JO); NIHGMS 5U54GM104942‐03 (PDC)

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 Transcriptomics and Functional Genetics Approach to Identifying Components of the Drosophila Polyamine Transport System
Authors: Saldanha Joanne , Haney Michael , Landry Brianne , Figueroa Natalia , von Kalm Laurence
Published: June 2021

Polyamines are essential growth factors for normal and malignant cells. Cells obtain polyamines via biosynthesis or transport from the extracellular environment. Polyamine depletion therapy is an attractive option to inhibit the growth of cancer cells. An FDA approved drug inhibits polyamine biosynthesis, however malignant cells typically respond by upregulating the polyamine transport system. Therefore, a dual therapy simultaneously targeting the biosynthetic and transport pathways is desirable. However, this approach is currently hampered by a poor understanding of the transport system in multicellular eukaryotes. To address this problem, we used RNA‐Seq to identify genes that are up‐ or down‐regulated under conditions where Drosophila larvae must transport polyamines to survive. From the screen 36 candidate genes for roles in polyamine transport were identified. We have also developed a functional genetic assay to test individual candidate genes. Our results suggest the existence of tissue‐specific polyamine transport systems, and post‐transcriptional regulation of polyamine transport.

Support or Funding Information

University of Central Florida, College of Sciences Seed 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.
Characterizing Insulin Sensitivity in Adult LEW.1WR1 Rats
Authors: Mercado Luis Daniel , Collins Genoah , Clopp Amelia , Love‐Rutledge Sharifa
Published: June 2021

Diabetes prevention is a large topic of research that is focused on nutritional and exercise‐based interventions with some genetic predisposition based interventions. FAT10 is a ubiquitin‐like protein and Type 1 Diabetes (T1D) susceptibility gene that may play a role in age‐related inflammation, adiposity, cancer, and kidney disease. The young T1D susceptible, LEW.1WR1 rat overexpresses FAT10 and has higher fasting concentrations of blood insulin. It is unclear how the overexpression of FAT10 effects directly affects insulin sensitivity, secretion, or production. It is also unclear if the initial insulin sensitivity of this animal model plays a role in disease susceptibility. Therefore we hypothesized that older LEW.1WR1 rats will have markers of increased insulin resistance like increased insulin and beta cell mass due to the poor regulation of FAT10 expression in this model. The objective of this study was to characterize insulin sensitivity, insulin levels, and characterize the relative beta cell size of adult LEW.1WR1 rats with or without dietary stress to understand if FAT10 expression plays a role in the regulation of inflammation and aging‐induced insulin resistance. To test this hypothesis we used LEW.1WR1 and LEW.SsNHsd rat and monitored weight gain and glucose tolerance over the course of 12 weeks on a high fat or control diet; we also isolated pancreata and used immunohistochemistry staining for insulin to analyze islet size and insulin staining. The LEW.ssNHsd rat served as a control rat because it does not have the alteration in the FAT10 promoter region. Glucose tolerance tests were performed prior to starting the diet as a baseline, and after 10 weeks on the diet to determine the effects of diet and aging on the rat groups in regards to Insulin Sensitivity. We observed that in the course of 10 weeks the control LEW.1WR1 rats became significantly more glucose intolerant and had increased insulin levels. The control LEW.1WR1 rats also gained weight at a rate similar to the high‐fat diet, a diet that is traditionally used to induce insulin resistance and glucose intolerance. This data suggests that FAT10 may be playing a role in age‐related adiposity, glucose intolerance, insulin sensitivity, and beta‐cell changes in the LEW.1WR1 rat. This study begins to lay the groundwork for understanding how alterations in FAT10 and metabolism increase T1D susceptibility.

Support or Funding Information

The University of Alabama in Huntsville

Louis Stokes Alliance for Minority Participation (NSF)

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 Noncanonical Role of Delta Protein Kinase C (δPKC) Phosphorylation of Troponin I in Cardiac Reperfusion Injury
Authors: Lin Amanda J. , Ostberg Nicolai P. , Kornfeld Opher , Qvit Nir , Mochly‐Rosen Daria
Published: June 2021

Injury of the myocardium due to reperfusion following an ischemic period (ischemia/reperfusion injury; IR) is the consequence of an imbalance between the production of oxidants and the availability of endogenous antioxidants. Depriving the heart of oxygen and nutrients (ischemia) leads to impaired mitochondrial function, elevated mitochondrial fission, cardiac damage, and death. Paradoxically, restoring blood flow (reperfusion) to the heart following an ischemic event poses another type of insult in the form of increased mitochondrial dysfunction, apoptosis, and necrosis, resulting in cardiac failure. Our laboratory has found that δPKC mediates reperfusion injury; treatment with a selective inhibitor of δPKC, δV1‐1, at reperfusion reduces infarct size by 60% in a variety of animal models. One of several substrates of δPKC, cardiac troponin I (cTnI), undergoes partial proteolysis upon phosphorylation by δPKC during IR. Interestingly, using western blot and microscopy studies, we found that the N‐terminal fragment of cTnI co‐localizes with mitochondria following IR and that inhibition of δPKC by δV1‐1 inhibits this cleavage. To determine the role of phosphorylated cTnI, we rationally designed a highly selective inhibitor of cTnI phosphorylation by δPKC, ψTnI. ψTnI inhibits only cTnI phosphorylation and not the phosphorylation of pyruvate dehydrogenase kinase (PDK), myristoylated alanine rich C kinase substrate (MARCKS), signal transducers and activators of transcription (STAT), and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) – substrates of δPKC that are elevated during reperfusion injury to the myocardium. ψTnI also diminishes cTnI association with mitochondria following reperfusion. Importantly, ψTnI treatment alone was sufficient to obviate acute IR injury in a whole heart ischemic model, reducing infarct size by over 70% and improving mitochondrial respiration when given at reperfusion. Additionally, ψTnI treatment at reperfusion of H9c2 cardiomyocytes restored mitochondrial oxygen consumption, preserved mitochondrial morphology, and rescued mitochondrial membrane potential in a dose‐dependent manner. We will determine the effects of cTnI phosphorylation and fragment association with mitochondria using loss‐ and gain‐of‐function mutations, live‐imaging, and functional assays. Together, our study identified a non‐contractile role for cTnI; in addition to regulating cardiac contractility, we find that cTnI regulates mitochondrial function following reperfusion injury to the heart.

Support or Funding Information

NIH grant R01‐HL052141 to DM‐R and AHA Postdoctoral Fellowship 19POST34380299 to AJL

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protective Effect of Intranasal Peste des Petits Ruminants Virus and Bacterine Vaccinations: Clinical, Haematological, Serological and Serum Oxidative Stress Changes in Challenged Goats
Authors: Emipke Benjamin Obukowho , Jarikre Theophillus Aghogho , Jeremiah Olalekan Adetokunbo , Akpavie Stephen Adetokunbo
Published: June 2021

The current vaccination for Peste des Petits ruminants PPR is stalled by myriad challenges and continuous endemicity of pneumonia due to fulminant bacteria complication in goats. The present study evaluated the protective effect of intranasal PPR virus linage 1 and bacterine vaccinations. Twelve West African Dwarf goats aged 6 months were randomly grouped and vaccinated within two weeks using combination of PPRV linage 1 vaccine (Nig/75), and bacterine from Mannheimia haemolytica (Mh) or Pasteurella. multocida (Pm) intranasally as A (vaccinated with PPRV & Mh), B (vaccinated with PPRV & Pm), and C (unvaccinated). All the goats were observed for three weeks post vaccination before comingling with a known infected WAD goat showing clinical signs of PPR and further observed clinically for five weeks (35days) post infection. Blood samples were taken for haematology and while sera were assayed for antioxidants (Glutathione Peroxidase GPx, Glutathione Transferase GST, Superoxide Dismutase SOD) activities and prooxidants (Malondialdehyde content, Reduced Glutathione GSH, Hydrogen peroxide H2O2 generation, Myeloperoxidase) using spectrophotometric methods. Data was subjected to parametric statistics at α=0.05 using Graph pad prism version 21. Clinically, there was pyrexia, mucopurulent oculo‐nasal discharge, diarrhoea, anaemia, leucopaenia and increased prooxidants in the unvaccinated goats (C), while moderate neutrophilia and leucocytosis were observed in PPRV and bacterine vaccinated goats. Two unvaccinated goats died at 13 and 28 days post infection. The goats vaccinated with PPRV and Mh showed better response. In conclusion, mucosal vaccination of goats with PPRV vaccine and bacterine will protect against exposure and culminate in the development of protective mucosal, humoral and cell‐mediated immune responses. This vaccination strategy will provide protection needed in the prevention and control of endemic caprine pneumonia in Nigeria.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
“Conditional Deletion of Hepatocellular Integrin Linked Kinase (hILK) Promotes an Increase in Hepatic Phosphoinositide 3‐kinase delta (PIK3Cδ)”
Authors: Martucci Nicole Jenna , Cotner Mary‐Clare , Bhushan Bharat , Mars Wendy M , Michalopoulos George K
Published: June 2021

Introduction

Biliary diseases accounted for over 100,000 deaths in the United States in 2016. When biliary cells (cholangiocytes) experience chronic injury during these diseases, hepatocytes can transdifferentiate into cholangiocytes to supplant the lost biliary cells. While the phenomena of transdifferentiation in the liver has been observed by many, the proteins and signaling pathways involved are not yet widely understood. Data from our lab has shown that with hepatocyte specific knockout of Integrin Linked Kinase (hILK KO), there is hepatocyte proliferation, increased matrix deposition, and an unorganized biliary cell/ductal proliferation; this led us to hypothesize that removal of hepatocellular ILK induces transdifferentiation of hepatocytes to cholangiocytes.

Methods

We originally generated hILK KO mice by repeatedly crossing mice expressing cre recombinase under an afp‐albumin promoter (Cre+/−) with mice that have floxed ILK genes (ILKfl/fl) to obtain mice that were ILKfl/fl:Cre+/− (hILK KO) and ILKfl/fl:Cre−/− (WT). To identify RNAs potentially involved in transdifferentiation, we performed data array analyses on livers from 7‐ and 14‐week old hILK KO and WT mice. RNAs of interest were then further examined by Western blot analyses and immunohistochemical staining.

Results

As anticipated from our previous published results, at 14 weeks, our data uncovered specific changes in RNAs related to matrix composition. A significant decrease was observed in Syndecans 1, 2 and 4 in the hILK KO mice. Collagen composition also changed as Col3a1, Col4a1, and Col1a4 and Col1a2 all increased in the hILK KO animals. Unexpectedly, our 7‐week RNA data analyses suggested a central role for Phosphoinositide 3‐kinase delta (PIK3Cδ) as it was commonly found in several of the Ingenuity Pathway Analysis pathways that were identified. PIK3Cδ is considered a cytoplasmic, leukocyte‐specific protein, however, in the hILK KO, the protein was also found in the nuclei of cholangiocytes and hepatocytes. pAKTS473, which is known to be downstream in PI3 kinase signaling pathways, and typically known as a plasma membrane protein, was also found in these nuclei, suggesting a direct relationship. We hypothesize nuclear PIK3Cδ is involved in the transdifferentiation of hepatocytes to biliary cells.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NF1 dissociates cell type specific contributions toward reward valuation and motor control
Authors: Sutton Laurie , Dao Maria , Brian Muntean , Martemyanov Kirill
Published: June 2021

Neurofibromin 1 (NF1) is a large multidomain signaling molecule that is a major Ras regulator but also acts as a positive regulator of cAMP levels by mediating G protein‐coupled receptor (GPCR)‐dependent activation of adenylate cyclase. Mutations in the NF1 gene cause Neurofibromatosis type 1, a genetic disorder characterized by multiple benign and malignant tumors with prominent neuropsychiatric symptoms that include learning and attention deficits, as well as motor impairments. Recently, we found that NF1 is a direct effector of GPCR signaling via Gβγ subunits in the striatum. However, it remains unclear the signaling contribution of NF1 in striatal‐mediator behaviors. Here we demonstrate the impact of intracellular signaling pathways commonly targeted by neurotransmitter inputs that impact medium spiny neurons (MSN) activity can be dissociated in a cell‐specific manner with distinct contribution to motor learning and reward‐related behaviors. We genetically ablated NF1 with cellular resolution demonstrating the diverging behavioral and signaling specific manner in which NF1 contributes to the acquisition and consolidating of learning a motor skill, as well as its regulation of morphine reward.

Support or Funding Information

This work was supported by the NIH and by the Canadian Institutes of Health Research (CIHR) Fellowship.

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 a time course for spontaneous opioid withdrawal in male and female outbred mice using an abbreviated dependence paradigm
Authors: Quock Raymond Mark , Brewer Abigail Lucille
Published: June 2021

Opioid withdrawal is often a limiting factor in recovery from opioid dependence due to negative reinforcement caused by continued use of opioids in order to avoid the distressing withdrawal symptoms. In animal models of dependence, opioid withdrawal is frequently precipitated using opioid antagonists and the severity of withdrawal is related to the level of dependence. However, this model lacks face validity when investigating new treatment possibilities for opioid withdrawal because most human addicts are not exposed to an antagonist in order to precipitate withdrawal. Furthermore, results from animal experiments suggest that spontaneous and precipitated withdrawal involve different mechanisms and may favor different symptoms of withdrawal. These experiments were conducted to investigate the timeline of spontaneous withdrawal in an outbred mouse model using an abbreviated dependence paradigm. Male and female NIH Swiss mice were injected with escalating doses of morphine twice daily (50–125 mg/kg morphine) over a period of four days. On the fifth day, mice were given a final dose of morphine and withdrawal symptoms were assessed at 5, 10, 24, 34, 48, 58, 72, 82, 96, and 106 hrs after the final injection. Withdrawal symptoms were assessed by global scores for an overall picture and by counting the number of tremors, wet dog shakes, and jumps. Results showed a robust withdrawal time course that peaked at 24 hrs in both sexes as assessed by global score. When broken down, wet dog shakes and tremors proved to be the most reliable indicator of severity of withdrawal and females showed more tremors than males for the 1st 24 hours of the time course. These results establish a spontaneous withdrawal time course in an outbred strain of mouse that shows that dependence can be reliably established over a four‐day period using fairly high escalating doses of morphine. Furthermore, these results suggest that individual symptoms of withdrawal should be assessed over time as well as global score.

Support or Funding Information

Supported in part by the State of Washington Initiative Measure No.171.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel sumanirole bivalent analogues as potent dopamine D2 receptor Go‐protein biased agonists
Authors: Yano Hideaki , Bonifazi Alessandro , Hoffman Alexander F , Lupica Carl R , Shi Lei R , Newman Amy H
Published: June 2021

The dopamine D2 receptor (D2R) is a major target for therapeutic development of treatments for neuropsychiatric disorders. In particular, D2R agonists have been used for neurological disorders such as restless leg syndrome and Parkinson's Disease. However, current D2R agonist pharmacotherapies have drawbacks including the development of tolerance and other adverse side‐effects. To potentially improve D2R agonist‐based medications, we have designed bivalent ligands that have two pharmacophores tethered by a link. This strategy aims to improve selectivity, affinity, and cell signaling activation bias. Indeed, it has been demonstrated that the secondary pharmacophore plays a key role in inducing activation bias towards either G protein, or β‐arrestin pathways.

Here we characterized a series of bivalent orthosteric‐arthosteric heteroaryl pharmacophores with assorted linkers and compared them with the potent orthosteric D2R‐selective agonist, sumanirole. Several bivalent analogues demonstrated modest increases in D2R affinity compared to sumanirole. D2R functional assays using bioluminescence resonance energy transfer (BRET) showed improved efficacy and potency for G protein coupling, but substantially diminished efficacy and potency for β‐arrestin recruitment. Analysis using an operational model of bias revealed significant G‐protein activation bias (>1000‐fold). Further, several compounds exhibited a G protein subtype (GoA over Gi1) activation bias. Finally, D2R‐mediated, G protein‐coupled inwardly‐rectifying potassium channel (GIRK) activity was measured in midbrain slices using electrophysiology. Recordings from D2R‐expressing dopaminergic cells in the ventral tegmental area confirmed prolonged GIRK activation by the biased bivalent compounds, compared to dopamine or sumanirole.

Our novel sumanirole bivalent analogues demonstrate a proof of concept for G protein activation bias and thus may lead to D2R agonist therapeutics with reduced tolerance and improved efficacy. With these novel tools, the preferential targeting and activation of Go‐proteins, the most predominant Gi‐like protein family in the brain, are now conceivable in animal models. This advance may aid in treatments of basal ganglia disorders with reductions in untoward side‐effects in humans.

Support or Funding Information

Work was supported by the Intramural funds of the National Institute on Drug Abuse

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gβ5‐R7 regulator of G protein signaling complex is a positive regulator of insulin secretion
Authors: Slepak Vladlen , Wang Qiang , Henry Taylor , Pronin Alexey
Published: June 2021

We present new data describing the function of the atypical G protein beta subunit Gβ5 (gene: Gnb5). Gβ5 is unique in that instead of Gγ dimerizes with regulator of G protein signaling proteins of R7 family (RGS6, 7, 9 and 11). These obligatory protein complexes are known to be highly expressed in neurons, but have also been found in other tissues, for example in endocrine glands. Here, we show that Gβ5‐R7 plays a novel and surprisingly important role in stimulated insulin exocytosis. We demonstrated earlier that CRISPR/Cas9‐mediated deletion of the Gnb5 gene ininsulin‐secreting cell line MIN6 results in a dramatic reduction insulin secretion induced by muscarinic M3 receptor (M3R). Our data on the knockout of Gnb5 in mice also results in about 2‐fold lower level of serum insulin, and in a dramatic reduction in M3R‐stimulated secretion occurs in Gnb5−/− primary islets. These results are unexpected because in other systems, the knockout of this RGS protein complex facilitates signal transduction, apparently to its GAP activity toward Gi family proteins. In this study, we show that deletion of Gβ5‐R7 strongly attenuates inputs from different secretagogues, not only M3R. The knockout of the Gnb5 gene affects Gq‐ and Gs‐mediated stimulation, but most surprisingly, there is a significant effect on glucose, the permissive factor for insulin release. The Gnb5 knockout does not impair second messengers cAMP, Ca2+and DAG in beta cells, but rather causes changes protein phosphorylation pattern. Our experiments also implicate regulation of some protein kinases including ERKs and ROCK and participation of cell‐to‐cell interaction mechanisms, possibly adhesion GPCRs. Thus, our experiments point to the existence of a novel function of the Gβ5‐R7 complex that may be not directly associated with its interaction with heterotrimeric G proteins.

Support or Funding Information

This work was supported by U.S. National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases Grants R01DK105427 (to V.Z.S.) and R01DK111538 (to Alejandro Caicedo). The authors declare no conflicts of interest.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Parathyroid‐targeted overexpression of Regulator of G‐Protein Signaling 5 (RGS5) causes hyperparathyroidism in transgenic mice
Authors: Balenga Nariman
Published: June 2021

The relationship between impaired calcium sensing, dysregulated parathyroid hormone (PTH) secretion, and parathyroid cell proliferation in parathyroid neoplasia is not understood. We previously reported that a GTPase activating protein, regulator of G‐protein signaling 5 (RGS5) is overexpressed in a subset of parathyroid tumors associated with primary hyperparathyroidism (PHPT), and that RGS5 can inhibit signaling from the calcium‐sensing receptor (CASR). In vivo, we found that RGS5‐null mice have abnormal PTH secretion. To gain a better understanding of the potential role of RGS5 overexpression in parathyroid neoplasia and PHPT and to investigate whether inhibition of CASR signaling can lead to parathyroid neoplasia, we created and characterized a transgenic mouse strain over‐expressing RGS5 specifically in the parathyroid gland. These mice develop hyperparathyroidism, bone changes reflective of elevated PTH, and parathyroid neoplasia. Further, expression of exogenous RGS5 in normal human parathyroid cells results in impaired signaling from CASR and negative feedback on PTH secretion. These results provide evidence supporting the setpoint hypothesis of parathyroid neoplasia and define a role for RGS5 in the pathogenesis of PHPT through inhibition of CASR signaling.

Support or Funding Information

PhRMA Foundation and American Cancer Society research grants.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Remodeling the interactions between TDP43 and RNA for development of therapeutics for ALS
Authors: Felemban Razaz A , François‐Moutal Liberty , Sayegh Melissa , Perez‐Miller Samantha , Gokhale Vijay , Zarnescu Daniela C. , Khanna May
Published: June 2021

Developing effective treatment strategies for amyotrophic lateral sclerosis (ALS), that affect upper and lower motor neuron, requires an understanding of the underlying cellular pathway that lead to motor neuron death and muscle atrophy. RNA dysregulation is a hypothesized disease mechanism in ALS. Studies have shown that pathological transactive response (TAR) DNA Binding Protein (TDP‐43) binds irreversibly to RNA in stress granules and forms membraneless prion‐like TDP‐43 aggregates in cytoplasmic motor neurons1. These membraneless compartment can form in cells through liquid‐liquid phase separation (LLPS). Recent study showed that mRNA secondary structure promotes LLPS to build membraneless compartments in cells2. Based on the TDP‐43. mRNA complex remodeling hypothesis by Coyne et al3, we hypothesized that remodeling the interactions between TDP43 and RNA using small molecules will decrease motor neuron toxicity. Using in silico docking, we screened 50,000 compounds on the RNA recognition motifs 1 and 2 of TDP43 (RRM1 and RRM2). We identified rTRD001 that was able to bind to TDP43 in RRM1 domain, disrupt TDP43 interaction with RNA, reduce cytoplasmic aggregate and cytotoxicity in motor neuron cell line NSC34, and mitigate TDP‐43 dependent phenotypes in an ALS fly model of TDP‐43 proteinopathy. Further studies are necessary in order to assess the mechanism and mode of action for this neuroprotective effect for this compound.

Support or Funding Information

‐This project was funded by a grant from NIH R01 NS091299 and ABRC/ADHS16‐162407 ‐ Scholarship funded by King said bin Abdulaziz university for health science, Jeddah, Saudi Arabia

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthesis of potential antioxidants by microwave‐assisted solid phase diazotations and Friedel‐Crafts reactions of hydroquinone
Authors: Metras Breanna Nichole , Torok Bela , Elstrom Clifford
Published: June 2021

Oxidative stress, a harmful condition, is due to an excess accumulation of free radicals in the body, leading to cellular damage and potentially pathological conditions. The damage caused by free radicals was connected to the aging process and numerous diseases such as cancer, cardiovascular diseases, neurodegenerative diseases (e.g. Alzheimer's and Parkinson's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS)) or macular degeneration. Antioxidants can scavenge reactive oxygen species and effectively reduce oxidative stress. Quinone/hydroquinone easily undergoes redox changes and is perceived as potential antioxidants. In this work we explored the synthesis of hydroquinone derivatives using a new microwave‐assisted heterogeneous catalytic diazotization and subsequent coupling of the diazonium salts with hydroquinone as well as by applying traditional solid acid catalyzed Friedel‐Crafts alkylation and diazotation reactions.

Support or Funding Information

Funding for this research was provided in part by the Sanofi Genzyme and Oracle Education Foundation grants to the College of Science and Mathematics

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Carbamate Derivatives of Colchicine Show Potent Activity toward Primary Acute Lymphoblastic Leukemia and Primary Breast Cancer Cells: In Vitro and Ex Vivo Study
Authors: Urbaniak Alicja , Jousheghany Fariba , Yuan Youzhong , Pina‐Oviedo Segio , Delgado Magdalena , Majcher Urszula , Klejborowska Greta , Huczyński Adam , Monzavi‐Karbassi Behjatolah , Kieber‐Emmons Thomas , Chambers Timothy C.
Published: June 2021

Colchicine (COL) is the major alkaloid isolated from the plant Colchicum autumnale. It shows strong therapeutic effects towards various types of cancer. Its biological activity is related to its ability to bind to tubulin causing microtubule depolymerization, mitotic arrest, and cell death. The colchicine binding site is an attractive target for potential tubulin polymerization destabilizers. Indeed, a large number of molecules interacting with the colchicine binding site have been designed and synthesized with significant structural diversity. The identification of new therapeutics based on COL structure is warranted due to the toxicity of COL towards normal cells. In order to increase potency and reduce toxicity against normal cells, while retaining the activities in the nanomolar range, a library of novel COL analogs, namely N‐carbamates of N‐deacetyl‐4‐(bromo/chloro/iodo)thiocolchicine, has been synthesized (Fig. 1).

COL and several of the derivatives arrested MCF‐7 cells in mitosis, and caused microtubule depolymerization. Compounds were then tested against two types of primary cancer cells; adult acute lymphoblastic leukemia (ALL) cells, and human breast cancer (BC) cells derived from newly excised human tumor tissue. These represent more clinically relevant drug screening models compared to established cell lines. Four novel colchicine derivatives showed higher activity towards primary ALL cells (IC50 = 1.1 ± 0.5 to 6.4 ± 1.4 nM) and nine were more potent towards primary BC cells (IC50 = 2.3 ± 0.0 to 10.3 ± 4.6 nM) compared to COL (IC50 = 8.6 ± 0.2 nM and 11.7 ± 3.1 nM, respectively) in cell viability assays. COL and two of the most active derivatives were also shown to be effective in killing BC cells when tested ex vivo using fresh human breast tumor explants derived from residual invasive ductal carcinoma and invasive mucinous carcinoma. The present findings indicate that the COL derivatives described here constitute promising lead compounds for targeting acute lymphoblastic leukemia and different subtypes of breast cancer.

Support or Funding Information

The present study was funded by a grant (to TCC) from the Arkansas Breast Cancer Research Program. Financial support by grant (to AH. UM, GK) of the Polish National Science Centre (NCN) – No. 2016/21/B/ST5/00111 is 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.
Polyphenol‐Rich Fraction of Parquetina nigrescens Quenches Dichlorvos‐Induced Cardiorenal Dysfunction through Reduction in Nitrotyrosine/ p38 MAPK pathways
Authors: Ogunpolu Blessing Seun , Ochigbo Grace , Oyagbemi Ademola Adetokunbo , Omobowale Temidayo Olutayo , Falayi Olufunke Olubunmi , Saba Adebowale Benard , Adedapo Adeolu Alex , Yakubu Momoh Audu
Published: June 2021

Parquetina nigrescens is commonly used to treat diseases in humans and animals in developing countries, including Nigeria. This study evaluates the effects of its polyphenol‐rich fraction (prf) on dichlorvos‐induced cardio‐ and renal toxicity. There were several factors assessed during this study, including cardiac and renal markers, serum myeloperoxidase and xanthine oxidase, and electrocardiograph (ECG) changes. The changes in electrocardiograph (ECG) were recorded. Immunohistochemistry of cardiac and renal p38 and nitrotyrosine was determined. Dichlorvos exposure caused a significant decrease in L‐glutathione (reduced glutathione) and other antioxidant enzymes with increases in malondialdehyde, myeloperoxidase, advanced oxidation protein products, and protein carbonyl levels. It also brought about alterations in microanatomy of the heart and kidneys accompanied by increases in serum creatinine and urea levels. Exposure to dichlorvos induced prolonged QRS interval and shortened QT durations in rats. Immunohistochemistry revealed lower expressions of cardiac nitrotyrosine and renal p38 (mitogen‐activated protein kinase; MAPK) in rats treated with prf of P. nigrescens. Combining all, prf of P. nigrescens demonstrated antioxidant as well as protective properties in the heart and kidneys of rats exposed to dichlorvos. It ameliorated dichlorvos‐induced cardio‐ and nephrotoxicity giving credence to its use in ethnomedicine.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Establishment of Optimum Conditions for Antioxidant Components Containing Curcuma L Longa (Turmeric) Leaf Extraction using Response Surface Methodology
Authors: Kim Sera , Ko Seok‐Chun , Kim Yoon‐Sook , Ha Sang‐Keun , Park Ho‐Young , Park Yong‐Gon , Lee Sang‐Hoon
Published: June 2021

Curcuma L longa (turmeric) is a functional material but its leaves are wasted as byproduct. This study focused on the possibility of turmeric leaves as a functional food material by establishing its extraction condition using a reflux condenser with water and different concentrations of ethanol. According to the results, water extraction was efficient for extracting its total phenolic compounds (2.741±0.099 μg/ml), flavonoids (4.776±0.010 μg/ml) within various temperature and time conditions. DPPH (486.78±12.73 μg/ml), ABTS (123.19±2.42 μg/ml), and H2O2 (849.06±23.07 μg/ml) radical scavenging activities were also high in water extraction. We used response surface methodology (RSM) to optimize extraction conditions for Curcuma L longa leaves. After establishing the extraction ratio, we applied a central composite design to identify the effects of independent variables, temperature (X1), time (X2) to dependent variables, yield (Y1). From these results, the optimal conditions were established as 150 minutes with a 1:25 ratio at 85°C, giving a 15.57% extraction yield. Using the leaf extract, we measured cell viability and cytokine production using Raw 264.7 cell line to confirm its anti‐inflammatory effects. We confirmed there is no cytotoxicity in Curcuma L longa leaf extract treat groups compared to lipopolysaccharide treat (control) group. Also, production of cytokines such as TNF‐α, MCP‐1 and IL‐1β in Curcuma L longa leaf extract treat groups, decreased comparing with control group. In this study, we confirmed the optimum extract conditions of turmeric leaves as fundamental data for its application in the industry. Furthermore, we provide a new insight on turmeric leaf extract as a health‐beneficial natural product by identifying its antioxidant and anti‐inflammatory properties.

Support or Funding Information

This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through High Value‐added Food Technology Development Program, funded by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) (117078‐03).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure‐Toxicity Relationships of Trans‐Cinnamic Aldehyde and Its Substituted Analogs
Authors: True Heather , Debusman Chris , Harrelson John , Stamper Brendan
Published: June 2021

Objective

Trans‐cinnamic aldehyde (CA) is a compound found in cinnamon and has promising potential as an agent for smoking cessation therapy. Current pharmaceutical options to treat nicotine addiction are only moderately effective and new approaches are needed. In this study, we aim to explore the hepatotoxicity profiles of CA and its structurally‐related analogs as one step in a more‐extensive evaluation of these agents to treat nicotine addiction.

Methods

Mouse hepatocytes were exposed to varying concentrations of CA or its structurally‐related analogs and toxicity was measured by determining median lethal dose (LC50) values. Stock solutions for all compounds were prepared in DMSO and proper vehicle controls were performed for all experiments. Cells were treated with either CA or one of eight analogs: α‐bromocinnamaldehyde (Br‐CA), α‐chlorocinnamaldehyde (Cl‐CA), 3‐phenylpropionaldehyde (sat‐CA), α‐methylcinnamaldehyde (Me‐CA), 2‐nitrocinnamaldehyde (N‐CA), 2‐trifluoromethylcinnamadehyde (CF3‐CA), 2,6‐difluorocinnamaldehyde (F‐CA), and 2‐methylcinnamaldehyde (2‐Me‐CA). After the LC50 determinations were completed, two different luminescence‐based assays were performed to assess reactive oxygen species production and glutathione (GSH) depletion during exposure to CA and its analogs. A cutoff of p < 0.05 was used to determine significance by means of one‐way analysis of variance (ANOVA) with post‐hoc Least Significant Difference (LSD) tests.

Results

Significant differences in LC50 values were observed across the tested compounds indicating minor structural changes to the structure of CA can have profound toxicologic effects. CA analogs modified to contain electron‐withdrawing groups, as well as CA itself, were generally more toxic (lower LC50 values) than those that did not contain electron‐withdrawing groups. Furthermore, this toxicity was associated with significantly greater reactive oxygen species production and GSH depletion compared to the other CA analogs at equi‐toxic doses.

Conclusion

These results indicate analogs that contain electron‐withdrawing groups share a similar or inferior toxicity profile compared to CA significantly impacting the generation of reactive oxygen species and GSH depletion. However, this toxicity profile is unique for other CA analogs that do not contain electron‐withdrawing groups, which appear to propagate toxicity through alternative cell death mechanisms. Information generated from this study will help to inform future pharmacologic studies in animal and human models as these agents are further evaluated as potential aids for smoking cessation therapy.

Support or Funding Information

This work was supported by the Pacific University Research Incentive Grant 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.
The CXCR7/CXCR4 heterodimer induced histone demethylation: A new mechanism of colorectal tumorigenesis
Authors: Qu Xianjun , Cui Shu Xiang
Published: June 2021

Purpose

Both chemokine receptors (CXCR) 7 and 4 are known to be involved in a vast array of pathological events in the development cancer. However, it is not yet known whether the CXCR7/CXCR4 heterodimer can form in vivo, and if it does, the functional difference between CXCR7 monomer and the CXCR7/CXCR4 heterodimer.

Experimental Design

The expression of two CXCRs, CXCR7/CXCR4 heterodimer and histone demethylase JMJD2A in human colorectal rectal cancer (CRC) tissue was detected by western blot and immunohistochemistry. We then studied the role of the CXCR7/CXCR4 heterodimer in CRC tumorigenesis in Villin‐CXCR7‐CXCR4 transgenic mice and APCMin/+/villin‐CXCR7‐CXCR4 mice.

Results

CXCR 7 and 4 can form heterodimers in vivo and promote colorectal tumorigenesis through histone demethylation. Compared with adjacent non‐neoplastic tissue, human colorectal rectal cancer (CRC) tissue showed a significantly higher expression of CXCR4 and CXCR7, which was co‐localized in the cancer cell epithelium. CXCR/CXCR4 heterodimerization was associated with increased histone demethylase JMJD2A. Villin‐CXCR7‐CXCR4 transgenic mice presented a greater degree of exacerbated colitis and tumorigenesis than villin‐CXCR7 and villin‐CXCR4 mice. The CXCR7/CXCR4 heterodimer also increased APC mutation‐driven colorectal tumorigenesis in APCMin/+/villin‐CXCR7‐CXCR4 mice. Further analysis showed that the CXCR7/CXCR4 heterodimer induced nuclear β‐arrestin 1 recruitment and histone demethylase JMJD2A, leading to histone demethylation and increased transcription of inflammatory factors and oncogenes.

Conclusion

Our data reveal a novel mechanism of colorectal tumorigenesis through CXCR7/CXCR4 heterodimer‐induced histone demethylation. Inhibition of CXCR7/CXCR4 heterodimer‐induced histone demethylation could be an effective strategy for cancer treatment.

Support or Funding Information

This work was supported by Natural Science Foundation of China (91629303)

The graphical abstract

image

The graphical abstract

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
TAK1 inhibitor as a potential sensitizer for chemotherapy in glioblastoma
Authors: Campolo Michela , Lanza Marika , Casili Giovanna , Paterniti Irene , Filippone Alessia , Caffo Maria , cardali Salvatore M , Cuzzocrea Salvatore , Esposito Emanuela
Published: June 2021

Glioblastoma (GBM) is the most deadly brain tumor, characterized by extremely poor therapeutic response and overall survival. Despite relevant progress in conventional treatments, mainly represented by the clinical use of Temozolomide (TMZ) as standard chemotherapy, the prognosis of such tumors remains almost invariably dismal. Transforming growth factor beta‐activated kinase 1 (TAK1) is an essential component in genotoxic stresses‐ induced NF‐κB activation and MAPK pathways; however, the role of TAK1 in the development of chemoresistance in GBM remains unknown. The aim of the study was to verify, in GBM human cell lines, the effect of TAK1 inhibition on the sensitivity of GBM cells to chemotherapy treatments. Moreover, we used GBM samples of TMZ‐treated patients to validate our its role. The human GBM cell lines U138, A172, U87 MG and L229 cultures were treated for 24h with 5Z‐7 ox and TMZ at a concentration of 5μM and 100μM respectively and then lysate for western blot analysis and comet assay. Moreover, glioblastoma tissues from patients treated with TMZ were processed for western blot analysis. Using GBM cell lines, we found that 5Z‐7‐oxozeaenol significantly augmented the cytotoxic effects of TMZ, blocking TMZ‐induced NF‐κB activation, reducing DNA damage and enhancing TMZ‐induced apoptosis in GMB cell lines. Moreover, our result showed a significant up‐regulation in TAK1, p‐p38, p‐JNK and NF‐κB in glioblastoma TMZ‐treated patients denoting the continuous involvement of TAK1/MAPKs in tumorigenesis following chemotherapy. Together, our results suggested the role of 5Z‐7‐oxozeaenol to increase the sensitivity of GBM cells to chemotherapy and to be an effective coadjuvant to current chemotherapeutic regimens, offering TAK1 inhibitors as a novel strategy into the cultivated fields of cancer research.

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.
Novel inhibitors of ABC transporters increase accumulation of transport substrates in multidrug resistant cancer cells and blood brain barrier cells
Authors: Nanayakkara Amila K. , Holcomb‐Webb Kasi L. , Bequeaith Beau A. , Aljowni Maha , Ammerman Lauren E. , Lippert Alexander R. , Vogel Pia D. , Wise John G.
Published: June 2021

Multidrug resistance (MDR) is a major cause of chemotherapy failure. Overexpression of ATP‐binding cassette (ABC) transporters, P‐glycoprotein (P‐gp) and breast cancer resistance protein (BCRP) are two well‐studied drug transporters which are associated with MDR. These two transporters also act as a major functional unit of the blood brain barrier to protect the brain from xenobiotics and toxins. Lack of clinically approved P‐gp and BCRP inhibitors renders chemotherapy treatments of many MDR cancers ineffective and obstructs drug uptake into the brain. Using computational methods, we previously identified a novel class of P‐gp inhibitors that were not transport substrates of the pump. Here we describe the effects of chemical variants of the previously identified P‐gp inhibitor, SMU‐29. The variants were generated using computational approaches or by structure‐based design to improve protein binding interactions. All variants showed improved efficacy in reversing paclitaxel resistance in the P‐gp over‐expressing DU145 TXR prostate cancer cell line and their ability of increasing P‐gp substrate calcein in these cells. Five of these variants were not transported by P‐gp and three of them were substrates of the pump. Four variants only affected P‐gp, but not BCRP function, making them more selective than the parental compound, SMU 29. The three variants that affected BCRP reversed MDR in a BCRP over‐expressing breast cancer cell line, MCF‐7 M100, and increased accumulation of BCRP substrates such as Hoechst 33342, mitoxantrone and daunorubicin in these cells, indicating the potential to be used as BCRP inhibitors. The variants that affected both P‐gp and BCRP also showed higher accumulation of P‐gp and BCRP substrates in a blood brain barrier model cell line, hCMEC/D3, showing the potential to open the blood brain barrier for increased drug uptake into the brain.

Support or Funding Information

This work was supported by NIH NIGMS [R15GM094771‐02] to PVD and JGW, SMU University Research Council, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas.

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