bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fluorescence Imaging Reveals a Potential Association of Laminin Receptor with SENP Machinery
Authors: Robinson Michael , Umbaugh Charles Samuel , Figueiredo Marxa Leao 
Published: November 2023

Laminin receptor (LR) is a protein overexpressed in cancer. LR contributes to tumor‐cell migration and proliferation, and restructures laminins to increase aggressiveness and gene expression of tumor cells. Thus, the abundance of the receptor is a reliable determinant of tumor severity. Little is understood about how LR enhances aggressiveness of tumor cells.To gain insight into the behavior of laminin receptor, we genetically fused a red fluorescent protein to LR (LR‐mCherry2). We confirmed the orientation and insertion of the gene via restriction digests. Using high‐throughput live cell imaging, we determined peak expression time and number of positively transfected cells for LR‐mCherry2 and mCherry.Because there is evidence that laminin receptor is associated with SUMO (Small Ubiquitin‐Like MOdifying) proteins, we explored the hypothesis that SUMO proteins and SUMO machinery were associated with LR in the cell. A second set of proteins, SENPs (SUMO‐Sentrin Specific Proteases), are responsible for removing SUMO when their addition is no longer required. We cotransfected PC‐3 cells with LR‐mCherry2 or mCherry2 and one of four EGFP fused SENPs (SENP 2, 3, 5, or 6).After live cell imaging, consecutive images were compiled into movies that revealed localization patterns for SENPs and LR‐mCherry2. Interestingly, we found that LR‐mCherry2 was expressed throughout the cell but it was strongly associated with SENPs in the nucleus. This suggests that the LR may interact with SUMO machinery in the nuclei of prostate cells.Support or Funding InformationSupported by Summer Research Program (SROP) (MR) and start up funds from the BMS department (MF).
Rapid progression of breast cancer cell proliferation in diabetes: Role of polyamines
Authors: Morwitzer Melody Jane , Chandra Surabhi 
Published: November 2023

Diabetes and cancer are comorbid conditions with a heavy toll on human life worldwide. Several cancer subtypes (pancreatic, breast, liver, and colorectal) rapidly progress to advanced stages and even metastasize in diabetic states. Though high blood glucose levels have been considered as fuel for growth of cancer cells, pathways leading to this condition are still under investigation. Cellular polyamines can modulate normal and cancer cell growth, and can be synthesized from multiple sources including endogenous amino acid precursors, diet and through degradation. Inhibition of polyamines can provide protection against diabetes in mouse models. It is not known however whether elevation of polyamines in diabetes is linked to cancer advancement. Thus, we hypothesized that high glucose stimulated polyamine production causes increased proliferation of breast cancer cells. Proliferation studies were performed with varying concentrations of glucose (5mM–25mM) treatment on MCF‐7 (early stage) and MDA‐MB‐231 cells (advanced stage) breast cancer cells. Though there was a significant increase in cell proliferation with HG (25mM) in both cell lines, MDA‐MB‐231 cells showed this effect at 72hr compared to longer incubation time with MCF7 cells (96–120hr). Inhibitor of polyamine synthesis (difluoromethylornithine, DFMO, 5mM) significantly suppressed cell proliferation observed with HG (25 mM). Protein levels of ornithine decarboxylase (ODC), the enzyme required for polyamine synthesis, was also elevated with HG treatment but the levels were restored to normal glucose (5mM) conditions upon treatment with DFMO. Our findings indicate that advanced stage breast cancer cells proliferate rapidly in hyperglycemic states compared to early stage breast cancer cells, and that polyamines are involved in the transition to advance stage of cancer.Support or Funding InformationUniversity of Nebraska‐Kearney start‐up funds (SC)
“Kill two birds with one stone”: Bioengineering of single RNA molecule to target miR‐21a and introduce miR‐34a in NSCLC cells
Authors: Petrek Hannah Elizabeth , Ho Pui Yan , Duan Zhijian , Batra Neelu , Jilek Joseph , Yu Ai‐Ming 
Published: November 2023

Lung cancer is the second most common cancer among both men and women in the United States. The majority of lung cancer cases are classified as non‐small cell lung cancer (NSCLC). Current NSCLC treatments include resection surgery, radiation, and pharmacotherapy which all provide some benefits and have limitations. As a result, lung cancer caused deaths account for about 27% of all cancer deaths in the US. Therefore, new therapeutic agents are highly demanded for NSCLC. MicroRNAs (miRNA or miR) are a class of genome‐derived, small noncoding RNAs in cells that govern target gene expression through mRNA degradation or translational inhibition. MiR‐34a is commonly downregulated in NSCLC and targets various oncogenes such as c‐MYC, c‐MET, BCL‐2 and SIRT. On the other hand, miR‐21a is upregulated in NSCLC and reduces the expression of many tumor suppressors such as PTEN and PDCD4. Aiming at targeting oncogene miR‐21a and introducing tumor suppressor miR‐34a simultaneously in NSCLC cells, we employed our recently developed RNA bioengineering technology to produce a single RNA molecule containing both miR‐34a and a siRNA against miR‐21a (si‐miR21a). Our data showed this si‐miR21a/miR‐34a agent was highly expressed in E. coli cells. After purification by FPLC method, si‐miR21a/miR‐34a agent was transfected into human NSCLC cells. We further defined the levels of cellular si‐miR21a, miR‐21a and miR‐34a levels, the effects on miR‐21a and miR‐34a target gene expression, and the impact on NSCLC cell proliferation. Using a single RNA agent for dual targeting represents a new strategy for the development of cancer therapeutics.Support or Funding InformationThis study was supported by grants R01GM133888 and U01CA175315 from NIH.
The interaction of GIV with Galpha‐i is a druggable protein‐protein interaction
Authors: DiGiacomo Vincent , de Opakua Alain Ibanez , Merino Nekane , Blanco‐Canosa Juan B. , Blanco Francisco J. , Garcia‐Marcos Mikel 
Published: November 2023

GIV (a.k.a Girdin) is a non‐receptor Guanine‐nucleotide Exchange Factor (GEF) for heterotrimeric G proteins. Dysregulation of signaling networks dependent on such G proteins is a contributing factor in many human pathologies from cancer to psychiatric disorders. Therapeutic development in this area has been very successful by targeting G protein‐coupled receptors (GPCRs) – the targets of greater than 30% of marketed drugs – but structural and mechanistic insights into regulators such as GIV have opened novel routes to pharmacological intervention. GIV itself is overexpressed in a variety of human cancers and promotes metastatic properties in tumor cells. Moreover, disrupting GIV binding to Gαi blunts this metastatic behavior. These findings situate the GIV‐Gαi interaction as a promising therapeutic target. Despite protein‐protein interactions (PPIs) generally being considered poorly “druggable,” here we provide structural details about the Gαi‐GIV interface and demonstrate its chemical tractability. In testing a collection of >1,000 bioactive compounds with two high‐throughput screening approaches (structure‐guided virtual screening and a fluorescence polarization assay), we discovered two common micromolar potency hits, which were confirmed as true inhibitors by orthogonal assays. We mapped the binding site of the top hit by NMR spectroscopy and biochemical assays, showing that it overlaps with the GIV engagement site on Gαi, thereby validating its mode of action. Moreover, this inhibitor did not block Gβγ binding to Gαi, indicating that GIV binding can be specifically inhibited. This work empirically confirms the Gαi‐GIV PPI as a druggable target and outlines a discovery approach for anti‐metastatic small molecules. We will present our progress to date in screening a larger library of 200,000 compounds.Support or Funding InformationFunded by NIH R01GM112631 and the Hartwell Foundation
Protective Role of Autophagy in NLRP3 Inflammasome Activation and Medial Thickening of Mouse Coronary Arteries
Authors: Bhat Owais , Meng Nan , Yuan Xinxu , Lohner Hannah , Li Pin‐Lan 
Published: November 2023

NLRP3 inflammasome is activated by proatherogenic stimulations to result in vascular injury, inflammation, and ultimately atherosclerosis. It remains unknown how this inflammasome activation is endogenously regulated during atherogenesis. The present study hypothesized that autophagy and associated lysosome function serve as a critical modulator in NLRP3 inflammasome activation upon proatherogeneic stimuli. We first detected inflammasome activation in isolated and cultured mouse coronary arterial smooth muscle cells (CASMCs) from wild type (CD38+/+) mice and mice with deletion of CD38 gene (CD38−/−), a gene encoding ADP‐ribosylcyclase that regulates lysosome function and autophagic flux. Western blot analysis showed that cleaved caspase‐1 was much higher in CASMCs from CD38−/− than CD38+/+ mice, and that increased active caspase‐1 led to enhanced IL‐1β release to the cell culture media. In animal experiments, we found increased colocalization of NLRP3 vs ASC or vs Casp‐1 as well as enhanced IL‐1β accumulation in the coronary arterial wall in CD38−/− mice on the Western diet (WD) compared to CD38+/+ mice. This WD‐induced NRLP3 inflammasome formation and activation could be blocked by treatment of rapamycin, an autophagy enhancer. However, chloroquine, a lysosme function inhibitor and blocker of autophagic flux, markedly enhanced WD‐induced NRLP3 inflammasome formation and activation. Morphological examinations confirmed that the media of coronary arteries was significantly thicker in CD38−/− mice on the WD than CD38+/+ mice. This WD‐induced coronary arterial thickening was completely blocked by treatment of mice with rapamycin but enhanced by chloroquine. In conclusion, deficiency of autophagic flux due to CD38 gene deletion promotes NLRP3 inflammasome formation and activation in CASMCs, and rapamycin‐induced autophagy reverses the effects of proatherogeneic stimulation via inhibition of the NLRP3 inflammasome. The efficient autophagy is essential in the control of NLRP3 inflamamsome activation preventing the medial thickening of coronary arteries in response to proatherogenic stimuli.Support or Funding InformationSupported by NIH grants HL057244, HL075316 and HL122937
Endothelial NLRP3 Inflammasome Activation and Neointima Formation in Mice by Gut Microbial Metabolite Trimethylamine‐N‐Oxide
Authors: Koka Saisudha , Li Pin‐Lan , Zhang Yang , Boini Krishna M 
Published: November 2023

Recent studies have demonstrated that trimethylamine‐N‐oxide (TMAO), an intestinal microbial metabolite induces atherosclerosis and enhances the risk for cardiovascular disorders. However, the precise mechanisms of action of TMAO in cardiovascular pathophysiology are yet to be elucidated. In the current study, we examined whether TMAO induces inflammasome activation and thereby contributing to endothelial dysfunction and vascular injury. Confocal microscopic analysis demonstrated the formation of Nlrp3 inflammasomes in the intima of the carotid arteries of Nlrp3+/+ mice but not in Nlrp3−/− mice. Correspondingly, TMAO‐induced increases in IL‐1β production in the carotid intima were abolished in Nlrp3−/− mice. The endothelial tight junction protein, ZO‐1 was reduced by TMAO in Nlrp3+/+ mice, but not in Nlrp3−/− mice. It was also found that TMAO significantly increased neointimal formation in partially ligated carotid arteries of Nlrp3+/+ mice, but not in Nlrp3−/− mice. In in vitro studies, we also found that TMAO treatment significantly increased the colocalization of NLRP3 with Asc or NLRP3 with caspase‐1 in endothelial cells compared to control cells. Pretreatment with Nlrp3 siRNA transfection abolished the TMAO‐induced inflammasome formation in these cells. Correspondingly, TMAO treatment significantly increased the caspase‐1 activity (2.2 folds) and IL‐1β production (1.8 folds) compared to control cells but not in Nlrp3 siRNA transfected cells. Furthermore, western blot analysis showed that TMAO treatment significantly decreased tight junction protein ZO‐1 level compared to control cells. In addition, TMAO treatment significantly increased the cell permeability of EOMA cell layers compared to control cells (Control: 1 ± 0.1 vs TMAO: 2 ± 0.07). Prior treatment with Nlrp3 siRNA attenuated the TMAO‐induced cell permeability. Together, these results suggest that TMAO‐induced formation and activation of NLRP3 inflammasomes in vascular endothelium thereby leading to endothelial dysfunction and atherosclerosis in mice.Support or Funding InformationDK104031
Differential Effects of Short Chain Fatty Acids on Endothelial NLRP3 Inflammasome Activation and Neointima Formation
Authors: Yuan Xinxu , Wang Lei , Bhat Owais , Lohner Hannah , Zhang Yang , Li Pin‐Lan 
Published: November 2023

Short‐chain fatty acids (SCFA) as a family of gut microbial metabolites have been reported to promote preservation of endothelial function and thereby exert anti‐atherosclerotic action preventing cardiovascular diseases. However, the precise mechanisms mediating this protective action of SCFA remain unknown. The present study investigated the effects of SCFAs (acetate, propionate and butyrate) on the activation of Nod‐like receptor pyrin domain 3 (NLRP3) inflammasome in endothelial cells (ECs) and associated carotid neointima formation. In cultured ECs (EOMA), we observed that butyrate significantly decreased the formation and activation of NLRP3 inflammasomes induced by 7‐ketocholesterol (7‐Ket) and cholesterol crystals (CHC) and that propionate only blocked CHC‐induced inflammasome activation. Acetate had no significant inhibitory effects on the NLRP3 inflammasome activation induced by either 7‐Ket or CHC, but itself even activated NLRP3 inflammsomes. Using a mouse model with partial left carotid ligation (PLCA), we demonstrated that butyrate significantly decreased NRLP3 inflammasome formation and activation in carotid arterial wall as shown by reduced co‐localization of inflammasome components (assembling) and lowered active caspase‐1 activity and IL‐1β level in wild type mice (ASC+/+), which was comparable to the inhibition of this inflammasome in mice with knockout of the adaptor protein apoptosis‐associated speck‐like protein gene (ACS−/−). Neverthless, both acetate and propionate markedly enhanced the formation and activation of NLRP3 inflammasome in carotid arteries with PLCA in ASC+/+, but not ASC−/− mice. Correspondingly, byturate suppressed, but acetate and propionate enhanced carotid neointima formation during PLCA in ASC+/+ mice. These results indicate that SCFAs have differential effects on endothelial NRLP3 inflammasome activation and associated carotid neointima formation.Support or Funding InformationSupported by NIH grants HL057244, HL075316 and HL122937
Glucosamine Hydrochloride and Tunicamycin Attenuate Palmitate‐Induced Glycosylation of Cyclooxygenase‐2 in Primary Human Vascular Smooth Muscle Cells
Authors: Raman Puneet , Tat Trinh , Gonzales Rayna J 
Published: November 2023

Cyclooxygenase‐2 is a well‐studied mediator of inflammation in the vessel wall, expressed minimally in normal physiological conditions but inducible in pathophysiological conditions like atherosclerosis. Increases in COX‐2 transcription result in prostanoid production, such as prostaglandin E2 (PGE2). PGE2 acts through multiple EP receptors in the vasculature to cause vasoconstriction, vasodilation, change vascular permeability, and promote remodeling. Both COX‐2 expression and PGE2 production contribute to the formation of unstable plaques and increased risk for thrombotic events. Recent studies have shown that free fatty acids such as palmitic acid can act as pro‐inflammatory ligands, but their effect on COX‐2 and regulation of vascular function and health is not well elucidated. Thus, the objective of the study was to investigate changes in post‐translational modification of COX‐2 in response to stimulation via palmitate, as well as pharmacological inhibition and the effect on downstream prostanoid production. Human vascular smooth muscle cells (HVSMC) of brain, aortic and coronary artery origin were treated chronically (12 to 18 hr) with vehicle (5% BSA and 0.1% ethanol), palmitate, tunicamycin, or glucosamine. Protein levels were assessed via immunoblotting and prostanoid production was assessed via ELISA. Palmitate increased the expression of COX‐2 and its glycosylation state. Co‐treatment with the N‐linked glycosylation inhibitors tunicamycin and glucosamine attenuated the increase in glycosylation state induced by palmitate. Additionally, palmitate‐induced increases in PGE2 production were attenuated by co‐treatment with tunicamycin. In conclusion, the results suggest that the increase of vascular COX‐2 glycosylation by saturated fatty acids increases COX‐2 activity, an effect attenuated by inhibition of glycosylation. This regulation of COX‐2 activity via post‐translational modification in the vasculature is a possible novel mechanism that may contribute to the development of cardiovascular disease by diminishing the health of vascular tissue.Support or Funding InformationValley Research Partnership (RG, PR) and University of Arizona Sarver Heart Center (PR)
Interaction between Sympathomimetic Amines, Norepinephrine, Tyramine and Dopamine at the Monoamine Transporter in Sympathetic Nerve Terminals and the Role of Nitric Oxide
Authors: Simaan Joseph 
Published: November 2023

We previously showed that fresh synthesis of nitric oxide (NO) is involved in the transport of sympathomimetic amines, norepinephrine, tyramine, ephedrine and mephentermine across the monoamine transporter (MAT) in sympathetic nerve terminals and also in the blockade of the transporter by cocaine, imipramine, methylphenidate and atomoxetine. This study explored the effect of NO on the transport of dopamine (DA) and compared it with its effect on the transport of norepinephrine (NE), directly–acting on adrenergic receptors, tyramine (T), indirectly–acting by releasing norepinephrine from vesicles in sympathetic nerve terminals and methoxamine (M), not a substrate of MA transporter. Synthesis of NO was blocked with Nw‐nitro‐L‐arginine (L‐NNA) and the rise in arterial pressure it induces was reduced to starting level by restoring the basal concentration of NO through an infusion of nitroglycerin, an NO donor, thus observing the effect of fresh synthesis of NO. Anesthetized Sprague‐Dawley rats were used in which mean arterial pressure was measured. The pressor effect of single doses of NE (0.05, 0.1, 0.2 μg) was potentiated by 44–23% (P< 0,000), of T (0.025, 0.05. 0.1 mg) was reduced by 31–23% (P<0.001) and of DA (5, 10, 15μg) was reduced by 47–41% (P<0.002). The pressor effect of M was not affected. It is concluded that fresh synthesis of NO is significantly involved in the transport of NE, T and DA across the MA transporter and that in the used doses, the pressor effect of DA is mainly due to its indirect effect by releasing NE from sympathetic nerve terminals. Supported by funds from the American University of Beirut.Support or Funding InformationSupported by funds from the American University of Beirut.
Deactivation of 12‐HETE by Peroxisomal β‐Oxidation by Alternatively Activated Macrophages
Authors: Kriska Tamas , Campbell William B. 
Published: November 2023

Polarization of macrophages to pro‐inflammatory M1 and to anti‐inflammatory or alternatively activated M2 state has important physiological implications in development of experimental hypertension and inflammation. 12‐ and 15‐hydroxyeicosatetraenoic acids (12‐ and 15‐HETE), the two major arachidonic acid metabolites of 12/15‐lipoxygenase, are essential in the process since the disruption of the encoding gene, Alox15, renders the mice unsusceptible to variety of types of experimental hypertension. The objective of this study was to test the hypothesis that alternatively activated M2 macrophages catabolize 12‐HETE into products that do not enhance angiotensin II‐mediated arterial constriction.We used cultures of M1‐ or M2‐polarized macrophages to study the metabolism of externally added [14C]‐12‐HETE. The 12‐HETE metabolites were separated by a reverse phase HPLC and identified by mass spectrometry.M2 polarized peritoneal macrophages converted most of the 12‐HETE into more polar metabolites within 2 hours of incubation. M1 macrophages, however, had little effect on the 12‐HETE catabolism. The same effect was observed in cultures of M1 or M2 polarized bone marrow‐derived macrophages. The major metabolite peaks were identified by mass‐spectrometry as β‐oxidation products. The conversion of 12‐HETE was inhibited by 50 μM thioridazine, a peroxisomal β‐oxidation inhibitor. Mitochondrial β‐oxidation inhibitors, 4‐pentenoic acid and etomoxir, had no effect on conversion. qPCR analysis of peroxisomal and mitochondrial β‐oxidation markers confirmed that only peroxisomal markers were upregulated upon M2 polarization. The major 12‐HETE metabolite peaks were collected and their activities were tested on abdominal aortic rings. While 12‐HETE (100 nM) pre‐incubation enhanced constrictions to angiotensin II (1–100 nM) from 15 % to 25 %, the metabolites lacked such an activity.Our results indicate that alternatively activated M2 macrophages are capable of degrading 12‐HETE and eliminating 12‐HETE's enhancement of angiotensin II‐induced vascular constriction, while M1 macrophages are not. These data support an antihypertensive role of M2 macrophages.Support or Funding InformationThis research was supported by the grant: NIH HL103673