bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of GSK3 beta and ERK in the human neutrophil response to fungal beta‐glucan (1046.5)
Authors: Johnson Courtney , Byrd Angel , Reichner Jonathan
Published: June 2021

Complement Receptor 3 (CR3), an integrin found on neutrophils, plays an important role in the neutrophil recognition and response to fungal components in the context of extracellular matrix proteins. Co‐ligation of CR3 with fibronectin (Fn) at the I‐domain and ß‐glucan (B) at the lectin‐like domain is possible due to spatially distinct locations and creates unique cellular activity not seen with ligation of either domain separately. Dual ligation of CR3 with Fn+B induces homotypic aggregation of primed neutrophils, a response representative of the immune cells encounter with fungi within tissues. To understand the signaling pathways involved in these CR3‐mediated responses, we employed various molecular techniques to determine the key proteins. Mass spectroscopy revealed a significant increase in tyrosine phosphorylation of nineteen proteins in human neutrophil samples exposed to FN+B but not in samples exposed to FN alone. Western blotting and immunopreciptation techniques validate these findings by showing an increase in two of the proteins, p‐ERK and p‐GSK. Further work using inhibitors UO126 (ERK inhibitor) and TZDZ‐8 (GSK3Beta inhibitor), show differing effects on cluster formation. Use of UO126 caused a complete abrogation of cluster formation and a significant loss of ERK phosphorylation. However, use of TZDZ‐8 caused a partial inhibition of cluster formation with little effect on GSK3Beta phosphorylation. Blotting for protein levels, we were able to show the phosphorylation of ERK increases over time while the phophorylation of GSK decreases. Use of TZDZ‐8 decreased the phosphorylation of ERK, while inhibition with UO126 had little effect on GSK3 phosphoryaltion. This result indicates a upstream role for GSK3 Beta in the phosphorylation of ERK. In addition, monoclonal antibody blocking of CR3 led to an increase in GSK3 Beta phosphorylation indicating CR3 signaling of p‐ERK may be regulated through GSK3 Beta phosphylation.

Grant Funding Source: Supported by NIH GM‐066194
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
In vitro 3D evaluation of penetrability of SRJ09, a semisynthetic anticancer agent (1048.4)
Authors: Wong Charng Choon , Periasamy Nagarajan , Sagineedu Sreenivasa , Sidik Shiran , Sumon Shariful , Phillips Roger , Lajis Nordin , Stanslas Johnson
Published: June 2021

The inability of anticancer drugs to penetrate through the tumor within their respective pharmacokinetic lifespan to destroy all the tumor cells is recognized as one of the factors that lead to poor outcome of chemotherapy. SRJ09 (3,19‐(2‐bromobenzylidene)andrographolide) has been identified as a lead anticancer agent on the basis of its selectivity towards breast and colon cancer cell line panels (Jada et al (2008) Br J Pharmacol, 155(5):641‐54). In our recent landmark study (Hocker et al (2013), PNAS, 110(25):10201‐6), we showed that the compound disrupts mutant K‐Ras activity by binding to it. In this present investigation, the penetrability of SRJ09 through the DLD‐1 colon cancer multi cell layer (MCL) was evaluated. DLD‐1 MCL is established by culturing the cells in collagen‐coated plastic insert. The amount of SRJ09 that penetrated through MCL was quantitated by utilizing high performance liquid chromatography (HPLC). Histopathological staining was used to visualize the morphology of MCL. Chemosensitivity assay was performed to assess the anticancer activity of SRJ09 in DLD‐1 cells. SRJ09 was able to penetrate through 3D tumor and is inversely proportional with the MCL thickness. Furthermore, the compound has better penetrability compared with doxorubicin, a clinical anticancer drug. Subsequent histopathological analysis revealed that the integrity of the MCL was retained and no visible damage was inflicted on the cell membrane, confirming the penetration of SRJ09 is by diffusion. Chemosensitivity assay demonstrated that SRJ09 was able to achieve IC50 value of 40.7 µM upon 1 hour exposure time on DLD‐1 cells which was approximately 4 folds lower than andrographolide, the parent compound of SRJ09. In conclusion, SRJ09 successfully penetrated through DLD‐1 MCL by diffusion and could be potentially developed as a clinically relevant anti‐colon cancer drug. This study was funded by Universiti Putra Malaysia Research University Grant Scheme (04‐01‐09‐0713RU; 04‐02‐12‐2017RU).
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.
Impact of lecturetools on student engagement and learning (1058.4)
Authors: Swanson Hollie , Hadley Robert , Piascik Michael
Published: June 2021

High student engagement is closely correlated with student success. This is not optimized in the traditional classroom utilizing lecture‐based delivery methods. The use of laptops and other mobile presents unique opportunities, such as when interfaced with the interactive polling software, LectureTools which allows for a creative, versatile two‐way interaction between student and instructor in real time. It also provides a rich source of data to be used for assessing individual and group performance. In this study, LectureTools was used by instructors for information delivery and the students for a specifically designed presentation. A questionnaire surveying students on their use of laptops and mobile devices in the classroom and the impact of LectureTools on learning outcomes was administered at the end of the semester. In addition, students were interviewed on a one‐on‐one basis. Results indicate that LectureTools is effective in facilitating student participation and enhancing their understanding of lecture content.
Alkaloid‐dependent fetal muscle‐type nicotinic acetylcholine receptor desensitization (1059.9)
Authors: Green Benedict , Lee Stephen , Welch Kevin
Published: June 2021

The exposure of a developing embryo or fetus to teratogenic alkaloids from plants has the potential to cause developmental defects in humans and animals due to the inhibition of fetal movement. The mechanism behind the inhibition of fetal movement is postulated to be the desensitization of fetal muscle type nicotinic acetylcholine receptors (nAChR). In this study, we compared the endogenous ligand acetylcholine (ACh), to coniine from poison hemlock (Conium maculatum) and nicotine from tobacco (Nicotiana tabacum) for the ability to desensitize fetal muscle‐type nAChR expressed by TE‐671 cells. The experiments were conducted using a membrane potential sensing dye‐based system and a two‐step agonist addition protocol. Treatment of the TE‐671 cells with 10 μM ACh reduced the subsequent dye response of the cells to 100 μM ACh by 72%. The sequential changes in membrane potential to nicotine and coniine were only reduced at 1 mM which was most likely due blockade of the nAChR ion channel by the agonist. This research was supported by USDA/ARS.
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
Autonomic dysfunction in veterans with Gulf War illness (1068.19)
Authors: Reyes Levy , Falvo Michael , Blatt Melissa , Ghobreal Bemin , Acosta Amanda , Serrador Jorge
Published: June 2021

Gulf War Illness (GWI) is a complex, idiopathic condition affecting Veterans deployed during the first Gulf War. Common symptoms reported in Veterans with GWI include but are not limited to chronic fatigue, diffuse muscle pain, gastrointestinal disorders, as well as autonomic dysfunction. While the cause of GWI is not known, there is evidence that autonomic dysfunction may be an important element underlying GWI and the associated disability. In this study Veterans with and without GWI (as defined by Kansas screening) underwent autonomic function testing, including transcranial Doppler in the middle (MCA) and anterior cerebral artery (ACA) as well as blood pressure during multiple tests (orthostatic challenge, hyper/ hypocapnia, cold pressor). Our results show that the autoregulation index (ARI) for both MCA (Control 3.5 ± 0.58 vs GWI 2.1 ± 0.48) and ACA (Control 3.7 ± 0.67 vs GWI 2.6 ± 0.50) are both lower in GWI Veterans vs. Non‐GWI vets; suggesting autonomic impairment in GWI individuals. Blood pressure nadirs did not change between groups (Control 9.9 ± 0.30s vs GWI 10.1 ± 0.32s); however, there was a difference when comparing nadirs from MCA mean blood velocity (7.6 ± 0.61s vs 8.9 ± 0.51s) between groups, further illustrating autonomic dysfunction in GWI Veterans. Our findings suggest a level of autonomic impairment in Veterans with GWI, and further studies should be performed to determine if interventions can ameliorate autonomic impairment in this group of individuals.

Grant Funding Source: Department of Veterans Affairs and Office of Public Health
Mapping the molecular dynamics of clathrin‐mediated endocytosis (107.3)
Authors: Merrifield Christien
Published: June 2021

Clathrin mediated endocytosis is the principle mechanism by which cells internalize cell surface receptors. It can be thought of as a molecular machine which concentrates receptors into patches at the plasma membrane and which bends the membrane into a vesicle that pinches off. Quite how the many structural, enzymatic and cytoskeletal components of the endocytic machine are spatially and temporally organised is not fully understood. In this talk I will describe our efforts to address this problem by analysing single endocytic events using total internal fluorescence microscopy (TIR‐FM).
Mitochondrial modulation of calcium pulsars in native endothelial cells (1075.3)
Authors: Béziau Delphine , Charbel Chimène , Toussaint Fanny , Blanchette Alexandre , Mayer Gaétan , Ledoux Jonathan
Published: June 2021

Calcium homeostasis is the crux of endothelial function and the regulation of vascular tone. Endothelial Ca2+ is thus highly dynamic and finely tuned. Amongst Ca2+ dynamics, Ca2+ pulsars are spontaneous Ca2+ release by IP3 receptors within myoendothelial projections (MEP). However, regulatory mechanisms of endothelial Ca2+ pulsars remain to be determined. Mitochondria play a major role in intracellular Ca2+ regulation in several cell types and may represent a modulator of endothelial Ca2+ dynamics like Ca2+ pulsars. We then hypothesized that mitochondria are active regulators of Ca2+ pulsars. Given the specific localization of Ca2+ pulsars, we first established the mitochondrial distribution in native mouse endothelium by confocal and electron microscopy. Mitochondria appeared heterogeneously distributed in endothelial cells but were found to be three times more concentrated near MEP. The functional interaction between mitochondria and Ca2+ pulsars was then investigated with real‐time confocal microscopy. Interestingly, mitochondrial uncouplers FCCP and CCCP significantly decreased Ca2+ pulsars frequency. Our data suggest that mitochondria are clustered near MEP to modulate Ca2+ pulsars and might therefore be an important regulator of endothelial function.

Grant Funding Source: Supported by FRQS, HSFC, CFI, SQHA and CIHR.
Nerve‐evoked contraction of isolated resistance arteries is modulated by protease‐activated receptors (1079.16)
Authors: Wei Ran , Zwozdesky Myron , Chen Michael , Plane Frances , Kerr Paul
Published: June 2021

The sympathetic nervous system is a key regulator of arterial diameter and thus blood flow and pressure. Protease‐activated receptors (PARs) are a novel class of G‐protein coupled receptors activated by proteolytic cleavage of the extracellular domain to reveal a tethered ligand. Four sub‐types of PARs have been identified, of which PAR‐1 and PAR‐2 are expressed in the vasculature. Activation of either PAR‐1 or PAR‐2 can evoke endothelium‐dependent relaxation but whether these receptors can modulate nerve‐mediated vascular responses has not been examined. We have investigated whether activators of PAR‐2 can modulate sympathetic nerve‐evoked increases in tone in rat isolated mesenteric resistance arteries.

In endothelium‐denuded segments of mesenteric artery, stimulation of peri‐arterial nerves (0.5 to 20 Hz) elicited frequency‐dependent increases in tone which were abolished by either prazosin, an α1‐adrenoceptor antagonist, or tetrodotoxin, an inhibitor of neuronal sodium channels, but were unaffected by depletion of sensory nerves with capsacin. Activation of PAR‐2 by either trypsin, the tethered ligand mimetic peptide fLIGRL or AC 264613, a synthetic PAR‐2 ligand, significantly attenuated nerve‐mediated responses. Increases in tone to bath applied noradrenaline were also attenuated by trypsin and fLIGRL but to a significantly lesser extent than the nerve‐evoked responses. The effects of trypsin on nerve‐evoked increases in tone were prevented by the trypsin inhibitor aprotinin. Thrombin, an activator of PAR‐1 also attenuated nerve‐evoked increases in tone although the effect was significantly less than that seen with PAR‐2 activation.

These data indicate that PARs directly modulate nerve‐mediated vasoconstriction of resistance arteries providing a novel mechanism for regulation of sympathetic vascular tone.

Supported by HSFC.
Sympathetic escape in a theoretical model of microvascular flow regulation (1079.18)
Authors: Roy Tuhin , Secomb Timothy
Published: June 2021

A mathematical simulation of flow regulation is used to investigate the hypothesis that vasoconstriction due to sympathetic nerve activity (SNA) in smaller arterioles is inhibited by functional vasodilation at higher values of oxygen demand, termed functional sympatholysis. A network with 13 vessel compartments in series is used, each compartment representing different size ranges of arterioles and venules. The model includes 5 actively regulating arteriolar segments with diameters influenced by shear stress, wall tension, metabolic regulation, and SNA. Metabolic signals are assumed to be propagated upstream along vessel walls via a conducted response. We show that for appropriately chosen parameter values, the vasoconstriction due to SNA in feed arterioles overcomes metabolic vasodilation at higher oxygen demand, resulting in persistent vasoconstriction throughout the period of SNA. In smaller arterioles under similar conditions, however, the initial vasoconstriction with SNA is followed by partial relaxation, consistent with the experimentally observed phenomenon of sympathetic escape. This implies that metabolic responses can account for observed characteristics of functional sympatholysis, including the different responses of large and small arterioles.

Grant Funding Source: Supported by NIH grant HL070657.
Impact of age and body position on the contribution of nitric oxide to femoral artery shear rate: implications for atherosclerosis (1079.21)
Authors: Trinity Joel , Groot H. , Layec Gwenael , Rossman Matthew , Ives Stephen , Richardson Russell
Published: June 2021

Age‐related alterations in shear rate in humans have only been examined in the atherosclerotic‐resistant conduit vessels of the arm. This study sought to examine the contribution of nitric oxide (NO) to age‐related alterations in shear rate and the impact of common body positions (supine and seated) in the atherosclerotic‐prone conduit artery of the leg. Inhibition of NO synthase (NOS) was accomplished by intra‐arterial infusion of L‐NMMA and common femoral artery diameter and blood velocity were measured by Doppler ultrasound in 8 young (24±1 yr) and 8 old (75±3 yr) healthy men. Old subjects exhibited reduced mean shear rate in the supine (18.2±2.5 sec‐1) and seated positions (17.2±2.5 sec‐1) compared with young subjects (supine: 41.5±5.9 sec‐1, seated: 32.2±4.0 sec‐1). This reduced mean shear in the old was driven by attenuated antegrade shear as there were no differences in retrograde shear. NOS inhibition reduced antegrade shear in the young such that age‐related differences were abolished. In contrast, NOS‐induced reductions in retrograde shear rate were similar between groups. The seated position reduced mean shear rate in the young to that normally observed in old. Overall, this study reveals that age‐related reductions in mean shear rate, assessed in the atherosclerotic‐prone vasculature of the leg, are largely explained by reductions in antegrade shear rate as a result of reduced NO bioavailability.

Grant Funding Source: Support by VA Advanced Fellowship in Geriatrics, VA Merit Award, and NIH PPG
Single‐molecule imaging of pre‐mRNA splicing in situ (108.3)
Authors: Sanjay Tyagi
Published: June 2021

We have developed a single‐molecule RNA imaging method to study the intracellular sites of splicing. In this approach introns and exons in natural genes are probed in situ with distinctively labeled sets of about 50 oligonucleotide probes. The attachment of so many probes renders each target molecule so intensely fluorescent that it becomes visible as a bright diffraction‐limited spot. Spots that fluoresce in just one color are either free introns or spliced mRNAs, and those that are visible in both colors correspond to pre‐mRNA molecules. Our imaging studies confirm that constitutively spliced introns are removed at the gene locus during transcription. However, during alternative splicing events regulated by RNA binding proteins Sex lethal (in fruit flies) and polypyrimidine tract binding protein (in HeLa cells), which ensure that only one splice form is produced in a particular cell type, splicing gets uncoupled from transcription and occurs after the release of pre‐mRNA from the gene locus. Similar uncoupling occurrs when intronic polypyrimidine tracts are masked within hairpins. Live cell imaging of the post‐transcriptional splicing events suggests that they occur at the periphery of nuclear speckles where many splicing factors and poly adenylated transcripts are sequestered. Recent genome‐wide studies reveal that a fraction of alternative splicing events likely occur via this route.
Targeting inflammatory serine proteases for cardiac repair after Ischemia Reperfusion injury (1080.10)
Authors: Hooshdaran Bahman , Kolpakov Mikhail , Rafiq Khadija , Guo Xinji , Kiani Mohammad , Sabri Abdelkarim
Published: June 2021

Background: Chronic inflammation predisposes tissue to adverse cardiac remodeling and contributes to heart failure (HF) progression. The mechanisms whereby inflammation affect cardiac remodeling after injury has largely focused on action of reactive oxygen species and cytokines/chemokines. However, inflammatory cells also release serine proteases and their role in myocyte loss and adverse cardiac remodeling remains largely unknown.

Methods and Results: We subjected mice to myocardial ischemia for 30 minutes followed by reperfusion for 24 hours (IRI) and assessed the effect of inflammatory serine protease (ISP) inhibitor, a potent non peptide inhibitor of cathepsin G, chymase and tryptase, on myocyte apoptosis, infarct size and cardiac function. ISP inhibitor was delivered just after reperfusion either systemically (10 mg/kg, i.v.) or encapsulated in anti‐P‐selectin conjugated immunoliposomes (2 mg/kg, i.v.). Treatment with ISP inhibitor loaded immunoliposomes reduced cathepsin G and chymase activity induced after IRI compared to mice treated with empty immunoliposomes. Targeted delivery of ISP inhibitor also reduced myocyte apoptosis, limited infarct size and improved cardiac function in response to IRI. These structural and functional improvements of targeted delivery of ISP inhibitor were similar to those obtained with systemic delivery of ISP inhibitor at higher dose, but have the advantage to occur without detectable side effects of ISP inhibition on spleen, bone marrow and circulating leukocytes.

Conclusions: These findings reveal the role of ISPs as key mediators of myocyte apoptosis and cardiac dysfunction post‐IRI and show that targeted inhibition of ISPs could be used as a therapy to reduce pathological cardiac remodeling and dysfunction associated with myocardial infarction.

Grant Funding Source: NIH R01360998
The energy regulating upstream kinase complex LKB1/MO25/STRAD is a potential novel regulator of thin filament function (1081.3)
Authors: Behunin Samantha , Lopez‐Pier Marissa , Konhilas John
Published: June 2021

AMP‐activated protein kinase (AMPK) is a cellular energetic regulator that is known to modify both metabolic and contractile function in the heart. Phosphorylation by an upstream kinase complex (LKB1/MO25/STRAD; LMS) is required for AMPK activation. Yet, the relationship between the LMS, AMPK activation, and myofilament function is unknown. Accordingly, we hypothesized that the upstream kinase complex can regulate myofilament function independently or in combination with AMPK. To do this, demembranated (skinned) rat cardiac trabeculae were incubated with either the LMS or with varying ratios of the LMS with AMPK and myofilament function was measured. Skinned rat trabeculae treated with the LMS alone were desensitized to Ca2+ and had lowered maximum tension. Adding increasing amounts of AMPK in combination with LMS reversed this effect increasing Ca2+‐sensitivity of tension and maximum tension. We furthered hypothesized that the mechanism underlying this relationship is mediated through direct interaction of the LMS with thin filament proteins. Western blot analysis showed an association of the LMS with myofibrils while immunofluorescence indicated that Mo25 co‐localizes with the phalloidin stained thin filament. In conclusion, the LMS has been identified as a potential novel regulator of myofilament function perhaps through a direct interaction with the thin filament.

Grant Funding Source: T32HL07249
Microvascular mechanisms of age‐related cognitive decline (1082.1)
Authors: Ungvari Zoltan , Toth Peter , Tucsek Zsuzsanna , Sosnowska Danuta , Gautam Tripti , Mitschelen Matthew , Tarantini Stefano , Deak Ferenc , Koller Akos , Sonntag William , Csiszar Anna
Published: June 2021

Hypertension in the elderly substantially contributes to cerebromicrovascular damage and promotes the development of vascular cognitive impairment. Despite the importance of the myogenic mechanism in cerebromicrovascular protection, it is not well understood how aging affects the functional adaptation of cerebral arteries to high blood pressure. Hypertension was induced in young (3 mo) and aged (24 mo) C57/BL6 mice by chronic infusion of angiotensin II. In young hypertensive mice, the range of cerebral blood flow autoregulation was extended to higher pressure values and the pressure‐induced tone of MCA was increased. In aged hypertensive mice autoregulation was markedly disrupted, and MCAs did not show adaptive increases in myogenic tone. In young mice the mechanism of adaptation to hypertension involved up‐regulation of the 20‐HETE/TRPC6 pathway and this mechanism was impaired in aged hypertensive mice. Downstream consequences of cerebrovascular autoregulatory dysfunction in aged angiotensin II‐induced hypertensive mice include exacerbated disruption of the blood‐brain barrier and neuroinflammation (microglia activation, up‐regulation of pro‐inflammatory cytokines and chemokines), which were associated with impaired hippocampal cognitive function. Collectively, aging impairs autoregulatory protection in the brain of mice with angiotensin II‐induced hypertension, potentially exacerbating cerebromicrovascular injury and neuroinflammation.

Grant Funding Source: Supported by the American Federation for Aging Research and the Nemzeti Fejlesztési Ügynökség
Suppression of cardio‐protective molecules by diabetic marker miR‐29: a mechanism for the high rate of cardiac damage in diabetes? (1086.6)
Authors: Arnold Nicholas , Koppula Purushotham , Pulakat Lakshmi
Published: June 2021

Diabetic marker miRNA‐29 (miR‐29) is up‐regulated by pro‐inflammatory cytokines and hyperglycemia. miR‐29 induced suppression of MCL‐1, a protein essential for mitochondrial function and cell survival, occurs in pancreatic β‐cell death. We hypothesized that conditions that increase miR‐29 levels in heart would promote suppression of MCL‐1 and increase cardiomyocyte cell death. Since Rapamycin (Rap) is known to promote diabetes, we further posited that Rap treatment (750 µg/kg/day; 6 weeks) would increase miR‐29 and suppress MCL‐1 in heart tissues of Zucker Diabetic Fatty (ZDF) rat, a rodent model for the progression of Type 2 Diabetes. Rap treatment attenuated plasma insulin (INS) levels implying development of insulinopenia; however, hyperglycemia persisted. q‐PCR showed a significant increase in miR‐29a, b, and c and suppression of MCL‐1 (p<0.01) in ZDF heart. Rap treatment also resulted in significant loss of cardiomyocytes as visualized by histopathological analysis by H & E and Masson's trichrome staining. Moreover, Rap treatment increased miR‐29 levels and suppressed MCL‐1 mRNA and protein levels in mouse atrial cardiomyocytes (10nM; 12hr) and in human coronary artery smooth muscle cells (20nM; 12hr). These data suggest that MCL‐1 regulation via miR‐29 family in INS‐responsive tissues is conserved across species and contributes to cardiomyocyte loss that can promote heart failure in diabetes.

Grant Funding Source: NIH: 1R01HL118376‐01