bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of expression profiles associated to oral squamous cell carcinoma development by cDNA microarrays (1047.13)
Authors: Oliveira‐Costa Joao Paulo , Gigliola Mabel , Carraro Dirce , Soares Fernando , Ribeiro‐Silva Alfredo 
Published: June 2021

Oral squamous cell carcinoma (OSCC) development still a poorly understood phenomenon, in terms of gene expression profiles which might be involved in alterations leading to high invasiveness and aggressive behavior. Also, it stills unclear how gene expression changes during OSCC development, in terms of tumor size. Using 36 samples of OSCC cases, we performed cDNA and lincRNA microarrays, comprising whole human genome and more than 7000 lincRNAs, in order to identify gene profiles with similar behavior throughout T1 to T4 tumor staging. Also, 10 differentially expressed genes were tested individually using qRT‐PCR. We found 7 different gene expression profiles, either with a rise in expression levels, or decrease in expression levels, between T1, T2, T3 and T4 TNM staging. The seven different gene expression profiles showed 58 differentially expressed genes, including an increased expression of immune response‐related family genes, and decreased expression of zinc finger proteins. In RT‐PCR experiments, we found significantly correlations between CD274 (PD‐L1) and tumor size (p=0.043), lymphatic invasion (p=0.047) and metastatic lymph node (p=0.001); BLNK and perineural invasion (p=0.021); ABL2 and tumor size (p=0.039); HOXB9 and tumor size (p=0.026); and between ZNF813 and tumor size (p=0.039). Our study shows the existence of several genes with similar expression patterns during OSCC development, and also points to novel genes, which has not yet been implicated in oral carcinogenesis, providing new potential targets in oral cancer research.Grant Funding Source: Supported by FAPESP grants 2010/08637‐0 and 2010/08400‐0
Identification of FOXO1 as a major transcriptional mediator of angiotensin‐(1‐7) signaling in tumoral cell lines (1047.3)
Authors: Passos‐Silva Danielle , Verano‐Braga Thiago , Peluso Augusto , Etelvino Gisele , Roepstorff Peter , Santos Robson 
Published: June 2021

Angiotensin‐(1‐7) has been shown to induce vasodilation, anti‐proliferative and anti‐tumorigenic activities via Mas receptor activation. However, little is known about the Ang‐(1‐7) signal transduction. We have shown using phosphoproteomics, that Ang‐(1‐7) fosforilates several kinases in human endothelial cells. In addition, it dephosphorilates the transcriptional factor FOXO1, which promotes its activation and nuclear translocation. When activated FOXO1 can increase transcription of genes involved in apoptosis, cell arrestment, angiogenesis modulation and tumor suppression. In this study we further address the consequences of FOXO1 activation by Ang‐1‐7 in tumoral cell lines. We observed that FOXO1 accumulated in the nucleus of A549 lung adenocarcinoma cells treated with Ang‐(1‐7), supporting the antitumorigenic activity of this peptide. Western blot results showed that CHO cells transfected with MAS receptor and treated with Ang‐1‐7 also showed the dephosphorylation of FOXO1, while ang1‐7 non‐transfected cells did not show this protein modification, indicating that this effect is mediated by MAS receptor. In addition, we treated tumoral cell lines such as A549 and DU145 with ang‐1‐7 in combination with different concentration of PI3K inhibitors, such as LY294002 or wortmannin. This treatment decreased the survival of these cells significantly in comparison to the treatment only with PI3K inhibitors. Thus, the combination of Ang‐1‐7 and PI3K inhibitors is a potential alternative treatment to cancer, since it involves the activation of different genes involved in tumor progression, angiogenesis and growth arrest.Grant Funding Source: INCT‐CNPq, CAPES, FAPEMIG
Acute Tocomin treatment improves endothelium‐dependent relaxation in aortae from diabetic and western diet fed rats. (1051.11)
Authors: Ali Saher , Nguyen Jason , Jenkins Trisha , Woodman Owen 
Published: June 2021

Tocotrienols, components of vitamin E, may have beneficial effects on vascular function in pathologies involving oxidant stress eg: diabetes. Tocomin (Toc) is an extract of palm oil with high tocotrienol content. We investigated the effect of acute Toc (10‐4 mg/mL) treatment on endothelium‐dependent relaxation (EDR) and superoxide (O2‐) production in aortae from diabetic rats and rats fed a standard (SD) or western (WD) diet. Wistar rats were treated with vehicle (control (C)) or streptozotocin (50 mg/kg iv) to induce diabetes (D) for 10 weeks. Wistar Hooded rats were fed SD (7% total fat) or WD (21% total fat). Rats were fed ad lib for 7 weeks after which they were placed on a restricted diet (70% of normal consumption for 10 weeks). EDR and O2‐ were measured using standard organ bath techniques and enhanced chemiluminescence. Diabetes impaired EDR which improved with Toc treatment (ACh Rmax C 855% vs D 644% vs D+T 873%, p0.05), whereas WD reduced ACh sensitivity that improved with Toc treatment (ACh pEC50 SD 7.200.14 vs WD 6.800.09 vs WD+T 7.270.12, p0.05). O2‐ production was increased in the diabetic and WD rat aortae and decreased with Toc treatment (C 57375 vs D 1053111 vs D+T 52858 and SD 518125 vs WD 953114 vs WD+T 31277 O2‐ counts/mg dry tissue, p0.05). Acute tocomin treatment improves EDR associated with decreased O2‐ production in aortae from diabetic and WD rats.Grant Funding Source: Nutricia Foundation
Spatiotemporally controlled delivery of biomolecules (1053.5)
Authors: Morales Demosthenes , Braun Gary , Pallaoro Alessia , Chen Renwei , Huang Xiao , Zasadzinski Joeseph , Reich Norbert 
Published: June 2021

Plasmonic nanoparticles are appealing for biomolecule and drug delivery due to their unique photophysical properties that provide spatiotemporally controlled release capabilities. Emerging techniques using light to control cellular functions, such as in optogenetics, motivates the design of novel methods to achieve mechanical manipulation of the cell. Here we demonstrate the release of a recombinant green fluorescent protein fused with a poly‐histidine tag to facilitate attachment to ~ 40 nm hollow gold nanoshells by nickel‐nitrilotriacetic acid affinity as well as a C‐terminal targeting peptide to promote cellular uptake of the nanoparticle‐protein composite. Laser release of protein was characterized by fluorescence for both particle and cellular studies, which was found to be power and time dependent. The construct presented may serve as a platform for further studies of protein delivery using nanoparticles.Grant Funding Source: This work was supported by National Institutes of Health (NIH) grant R01 EB012637.
Characterizing the abuse potential of loperamide via physiologically‐based pharmacokinetic/pharmacodynamic modeling and simulation (1053.6)
Authors: Ainslie Garrett , Kharasch Evan , Pollack Gary , Paine Mary 
Published: June 2021

Increased restrictions on opioid prescribing may fuel alternate means of abuse. Anecdotal reports suggest abuse of the over‐the‐counter opioid loperamide when taken at supratherapeutic doses with CYP3A/P‐gp inhibitors. As clinical assessment would be difficult, a CNS‐effective loperamide dose was determined via physiologically‐based pharmacokinetic (PBPK)/pharmacodynamic (PD) modeling and simulation. Alfentanil was used as a test opioid due to a straightforward plasma concentration‐effect relationship and rich clinical PK/PD data. An alfentanil PBPK model was developed using in vitro literature data and an absorption rate constant recovered from data obtained from 10 subjects administered alfentanil 1.7 mg po; the model was evaluated with the po clinical data. Alfentanil Emax (6.2 mm; CV, 43%) was recovered by fitting a sigmoidal model to iv plasma concentration‐miosis data with fixed PD parameters (EC50,brain,unbound, Hill slope) from mice. Miosis‐time profiles were simulated for 10 subjects. Geometric mean [90% CI] peak miosis (1.3 [0.8‐2.2] mm) and area under the effect curve (1.9 [0.9‐4.4] mm‐h) agreed with po clinical data (2.0 [1.6‐2.5] mm and 2.3 [1.7‐3.1] mm‐h, respectively). A loperamide PBPK/PD model was developed using an existing PBPK model, human alfentanil Emax, and mouse loperamide PD parameters. Simulations indicated a loperamide dose of 68 mg would achieve a miotic effect equivalent to an alfentanil po dose of 1.7 mg. This PBPK/PD model can be used to assess the impact of concomitant CYP3A/P‐gp inhibitors on the abuse potential of loperamide.Grant Funding Source: Supported by NIH R01 GM077482 & DA014211, Garrett R. Ainslie was supported by NIH T32ES007126
An in silico pharmacokinetic/pharmacodynamic model of agonist self‐administration behavior in rats (1053.8)
Authors: Ross Alexander , Tsibulsky Vladimir , Norman Andrew 
Published: June 2021

Rats trained to maintain self‐administration of dopamine (DA) receptor agonists do so with mathematically predictable regularity. The goal of this research is to model a proposed receptor‐based mechanism underlying the satiety threshold model of maintained self‐administration. MATLAB Simbiology was used to construct a multi‐compartment pharmacokinetic/pharmacodynamic model with a receptor population that exhibits mutually exclusive binding with either an agonist or a competitive antagonist according to the law of mass action. This model also incorporates drug distribution and elimination rate constants. Self‐administration events are programmed to occur only if the amount of agonist‐receptor complexes is at or below a threshold above which no further self‐administration events occur. Simulations show that the inter‐event interval is proportional to the unit dose, competitive antagonists decrease the inter‐event interval until they are eliminated from the system, and reducing the population of receptors via modeling irreversible antagonism also decreases the inter‐event interval. These simulations are consistent with maintained self‐administration behavior in rats, and suggest that the proposed receptor‐based mechanism is a viable explanation for this behavior. This model provides a useful tool for investigating the potential of antagonist‐based pharmacotherapies for drug addiction.Grant Funding Source: NIH Grant DP1DA031386
Discovery and characterization of the first cellularly active, non‐covalent inhibitor of a regulator of G protein signaling protein (RGS protein) (1054.10)
Authors: Mackie Duncan , Bodle Christopher , Hayes Michael , Roman David 
Published: June 2021

Regulator of G‐Protein Signaling (RGS) proteins are known to regulate the complex signaling pathways activated by GPCRs. Recent studies have implicated RGS proteins in the development and progression of pathologies, including some cancers. Over the past 10 years there have been several attempts to modulate these RGS proteins with all reported compounds working through a covalent Cysteine modification. These attempts have proven useful and laid the foundation for successful targeting of RGS proteins.Recently, our lab has focused on RGS17. This protein has been implicated in the growth, proliferation and metastasis of prostate cancer as well as small‐cell and non‐small cell lung cancers. We implemented a novel high‐throughput screening campaign to determine the first Gαo: RGS17 protein: protein interaction (ppi) inhibitors. This campaign yielded three compounds (UI5, UI1590 and UI1956) that inhibited the Gαo: RGS17 ppi in both biochemical and activity assays, while exhibiting single digit μΜ to nM dissociation constants by Isothermal Titration Calorimetry. These newly discovered inhibitors have cellular activity, disrupting RGS17 membrane localization in HEK293 cells down to 10 μM. Most importantly, UI1956 was specific for RGS17, as it had no effect on the ppi of RGS4 and RGS8 when incubated with Gαo. Finally, UI5, UI1590 and UI1956 did not covalently modify RGS17 as determined by mass spectrometry. Research is supported by American Foundation for Pharmaceutical Education (AFPE), The University of Iowa’s Center for Biocatalysis and Bioprocessing (5T32GM008365‐23) and The National Institutes of Health (R01CA160470).
Prevalence of inactivating protein kinase C mutations in human cancers (1055.2)
Authors: Antal Corina , Kang Emily , Stephenson Natalie , Trotter Eleanor , Hunter Tony , Brognard John , Newton Alexandra 
Published: June 2021

Protein kinase C (PKC) levels are aberrant in diverse cancers and over 400 mutations in PKC have been identified in human cancers. Here we characterized PKC mutations identified in human cancers to investigate whether PKCs function as oncogenes or tumor suppressors or whether their function is isozyme and/or context dependent. Live‐cell imaging was used to determine how these mutations affect PKC translocation, subcellular localization, and activity. Cell‐based assays were used to determine how PKC mutations alter proliferation, apoptosis, migration, and anchorage‐independent growth. Of 37 mutations we examined thus far, 24 of them reduced or completely abolished PKC activity, while only 1 increased it. These mutations reside within the diacylglycerol‐binding C1 domain, the Ca2+‐binding C2 domain, or the kinase domain of conventional, novel, or atypical PKCs. The inactivating mutations ablate activity by either impeding Ca2+ or diacylglycerol binding, or by yielding a misfolded kinase. Our results suggest that PKC can act as a tumor suppressor in certain cancers. Understanding whether mutations are activating or inactivating in specific cancers would allow us to determine whether therapeutic strategies should target ways to inactivate or activate PKC in the respective cancers.Grant Funding Source: This work was supported by GM43154 to ACN and DGE1144086 and T32 GM007752 to CEA.
Resistance of store‐operated calcium entry to tumour microenvironment conditions and enhanced potency of Synta66 in colorectal adenocarcinoma cells (1057.4)
Authors: Appleby Hollie , Gaunt Hannah , Jansz Sabrina , Hyman Adam , Stokes Ollie , Young Richard , Foster Richard , Beech David 
Published: June 2021

Store‐operated CRAC (SOC) channels have been suggested as drivers of tumour expansion. The aim of this study was to determine the effectiveness of a putatively specific SOC channel inhibitor (Synta66) in tumour microenvironment conditions. Cytoplasmic calcium concentration in HT‐29 colorectal adenocarcinoma cells was measured using a 96‐well plate fluorometer and the calcium indicator fura‐2. Hydrogen peroxide (1 µM‐1 mM), arachidonic acid (1 µM‐50 µM) and pH range 6.4‐7.4 were used as examples of common tumour microenvironment conditions. Contrary to expectations from studies of other cell types, hydrogen peroxide and arachidonic acid had no effect on SOC‐mediated calcium entry. pH 6.8 caused partial inhibition. The calcium entry was readily detected in the combined presence of all three conditions. Synta66 inhibited the calcium entry and was more potent in the presence of hydrogen peroxide, arachidonic acid or pH 6.8. The data support the hypothesis that chemical inhibitors of SOC channels may be useful for tumour suppression.Grant Funding Source: Supported by the British Heart Foundation, Cancer Research UK, and the Medical Research Council.
Polar substitutions in the ion‐conducting pore of GLIC alter gating and alcohol modulation (1061.9)
Authors: Facey Jody‐Ann , Venner Laura , Hyde Michael , Pouya Iman , Lindahl Erik , Howard Rebecca 
Published: June 2021

Pentameric ligand‐gated ion channels in the cys‐loop receptor superfamily mediate direct modulation of neurological function by alcohol. However, the absence of high‐resolution structures of human cys‐loop receptors has restricted our understanding of this process at the molecular level. Recent X‐ray crystallographic studies have revealed a binding site for alcohol near the ion‐conducting pore of GLIC, a bacterial cys‐loop receptor homolog, which suggests that alcohol might be involved in the opening and closing of the channel. To test this prediction, we generated site‐directed mutations in GLIC and measured their effects on ion channel gating and alcohol modulation by two‐electrode voltage clamp electrophysiology in Xenopus laevis oocytes. Wild‐type GLIC receptors exhibited gating and modulation properties in keeping with published literature, as did a mutant form associated with enhanced alcohol sensitivity. Polar amino acid substitutions at pore‐lining residues enhanced ion channel gating but reduced alcohol potentiation, both in the context of the wild‐type and alcohol‐sensitized variant. These results are consistent with mechanistic predictions from microsecond‐scale molecular dynamics simulations. Continuing characterization of pore substitutions in GLIC will further substantiate this system as a model for structure and function of cys‐loop receptors, and clarify our mechanistic understanding of alcohol modulation at the molecular level.Grant Funding Source: Supported by grants VR 2010‐491, ERC 209825, and by startup funds from Skidmore College.
Structure‐function analysis of a small‐molecule binding site in Kir1.1 and Kir7.1 (1062.4)
Authors: Kharade Sujay , Denton Jerod 
Published: June 2021

Inward rectifier potassium (Kir) channels play fundamental roles in diverse physiological processes and may be unexploited drug targets. However, the limited pharmacology has slowed efforts to explore the physiology and druggability of most Kir channels. We discovered a small‐molecule termed VU590 that inhibits Kir1.1 and Kir7.1 with IC50 values of ~300 nM and ~8 µM, respectively, but not Kir2.1 or Kir4.1. Kir1.1 regulates renal tubule salt and water transport and is an emerging diuretic target for the treatment of hypertension, whereas the functions of Kir7.1 are largely unknown. The aim of this study is to identify structural elements in Kir1.1 and Kir7.1 that imbue these channels with selective sensitivity to VU590. Patch clamp analysis revealed that VU590 can be displaced from the pore with inwardly directed K+ ions, suggesting the binding site is located in the membrane‐spanning pore of the channel. Indeed, mutation of Asn171 to the corresponding residue in Kir2.1 (Asp) caused a ~20‐fold loss of potency and increased the voltage‐dependence of block. We are currently expanding our mutagenesis analysis of this region in Kir1.1 and testing whether corresponding residues in Kir7.1 are required for VU590 sensitivity. These studies will provide novel insights into the structure of the Kir channel pore, in general, and inform future efforts to develop more specific inhibitors of Kir1.1 and Kir7.1.Grant Funding Source: Supported by NIH 1RO1DK082884 to JSD
The effect of glutamine, glutathione and glutamine plus glutathione as substrates for the preservation of the isolated perfused rabbit kidney (1063.3)
Authors: Fonteles Manassés , Sousa‐Filho José , Nascimento Nilberto Robson , Araújo‐Filho Raimundo 
Published: June 2021

Rabbit kidneys were perfused with a high K+ and Mg++ solution as previously described (J S R 21(3);169,1976), with addition of substrates. Briefly the animals were anesthetized with urethane and sodium pentobarbital and the left kidney perfused at 37ºC with a previously dialyzed solution (4g% albumin) in an artificial heart‐lung machine. Several functional physiological parameters were evaluated such as perfusate and urine flow, GFR, %TNa, %TK, perfusion pressure and vascular resistance. The experiments lasted 2 hours. To measure kidney function creatinine was added to the perfusate and Na,K, and osmolarities were measured every ten minutes. Urinary flow increased 2.5‐fold in the Glutamine group when compared to control. This parameter increased 3 times in the Glutathione group and 2.1‐fold in the Glutamine + Glutathione group when compared to control. The same trend was observed with GFR that was enhanced 2.3 times in the Glutamine group, 4.1 times in the Glutathione group and 3 times in the Glutamine + Glutathione group. The fractional sodium reabsorption increased 5.0% with Glutamine, 13.0% with Glutathione and 17.0% with Glutamine + Glutathione. No changes were observed in the fractional transport of potassium. We conclude that these 2 substrates, isolated or combined, promote a great improvement in kidney function in this ex vivo model. These substrates could be important metabolic support when perfusing kidneys for transplantation.Grant Funding Source: CNPq/Brazil
Human bronchiolar epithelial cells functionally express P‐glycoprotein and multidrug resistance‐related protein‐1 (1064.11)
Authors: Muchitsch Viktoria , Gausterer Julia , Salomon Johanna , Ehrhardt Carsten 
Published: June 2021

The human bronchiolar epithelial cell line, NCI‐H441 has recently been introduced as an in vitro model for studying drug disposition at the distal lung air‐blood barrier. Here, we determined levels of expression and function of major ATP‐binding cassette transporters, P‐glycoprotein (P‐gp) and multidrug resistance‐related protein‐1 (MRP1) in NCI‐H441 cells.P‐gp and MRP1 expression was quantified by immunoblot, whilst confocal laser scanning microscopy (CLSM) was employed to determine transporter localisation. Efflux experiments with rhodamin 123 (Rh123, P‐gp substrate) and 5,6‐carboxyfluorescein‐diacetate (CF, MRP1 substrate) were carried out assessing transporter function in vitro. Furthermore, the inhibitory effects of LY‐335979 (specific P‐gp inhibitor) and MK‐571 (specific MRP1 inhibitor) on Rh123 and CF efflux were investigated.Immunoblot provided evidence that NCI‐H441 cell monolayers expressed both efflux pumps on protein level. CLSM data confirmed these observations and moreover, revealed localisation of the transporters along apical membranes and in perimembranous vesicles. Rh123 and CF were both released from NCI‐H441 monolayers in a time‐dependent manner. This efflux could be inhibited by the relevant pharmacological agents.
Exploring the role of the unique N‐termini of PDE4D variants in regulating mural cell morphology, migration and cAMP signaling (1065.1)
Authors: Freitag Silja , Truong Tammy , Brzezinska Paulina , Umaña M. , Rampersad Sarah , Wudwud Alie , Hubert Fabien , Maurice Donald 
Published: June 2021

Mural cells act adaptively to regulate vascular tone. After vascular damage, adoption of a synthetic phenotype increases proliferative and migratory capacity and aids in repair. Exaggerated, maladaptive responses can promote intimal hyperplasia, atherosclerotic lesion progression and arterial (re)stenosis. cAMP acting on protein kinase A (PKA) or exchange protein activated by cAMP (EPAC) regulate mural cell migration and adhesion. A cAMP signalosome in the leading edge of migrating mural cells contains PKA and phosphodiesterase (PDE) 4D8 and regulates actin assembly and migration. PDE4D isoforms share a common catalytic domain and C‐terminus. PDE4 inhibitors are non‐selective with respect to the individual PDE4 variants and compete with cAMP binding at the active site. The unique N‐termini of PDE4D isoforms are hypothesized to mediate selective protein‐protein based tethering to distinct subcellular domains. We examined the subcellular targeting, function and binding partners of the unique N‐termini of mural PDE4D variants and their impact on cAMP signalling. We show that these N‐terminal regions are important in regulating mural cell morphology, migration and adhesion.Grant Funding Source: Supported by Canadian Institutes of Health Research