bluuebunny/crossref_metadata_2025_split huggingface dataset:
Naturally Occurring Mutations in Muscle‐Type Creatine Kinase Impact Tenofovir Monophosphate Phosphorylation
Authors: Mosher Eric P. , Wade Herschel , Bumpus Namandje N. 
Published: June 2021

Tenofovir (TFV), administered as a tenofovir disoproxil fumarate prodrug, is a nucleotide analog reverse transcriptase inhibitor used in combination for the treatment and prevention of HIV. TFV requires two sequential phosphorylation events in order to become pharmacologically active. We previously found that in colon tissue, a putative site of HIV infection, the muscle‐type isoenzyme of creatine kinase (CKM) is responsible for the second TFV activating phosphorylation step, converting tenofovir monophosphate (TFV‐MP) to the active tenofovir diphosphate (TFV‐DP). Due to the similarity in structure between TFV‐MP and ADP, this is likely analogous to the endogenous activity of CKM, which regenerates ATP from ADP by phosphoryl transfer from phosphocreatine. In addition, we have previously discovered naturally occurring genetic variants of the CKM gene in humans. Some of these single nucleotide variants are predicted to cause single residue mutations in the translated protein, a fraction of which are predicted by bioinformatics programs to have deleterious effects on enzymatic function. In the present study, molecular docking was employed to investigate the potential effects of these mutations on TFV monophosphate (TFV‐MP) binding and phosphorylation by CKM. These results indicate CKM binds TFV‐MP similar to how it binds its natural substrate, ADP. Molecular docking simulation also predict a cluster of active site arginine residues at positions 130, 132, 236, 292, and 320 are likely important in the charge stabilization of the phosphates of TFV‐MP, which may impact substrate binding and phosphorylation. To directly investigate the impact of the aforementioned mutations on TFV‐MP phosphorylation, CKM wild type and mutant proteins were recombinantly expressed and purified. In vitro assays were then performed to compare the ability of the mutant proteins to convert TFV‐MP to TFV‐DP. Enzyme activity was measured by liquid chromatography‐tandem mass spectrometry using a method that separated and detected the phosphorylated tenofovir metabolites concomitantly. It was found that R130H and R132C mutations decreased enzymatic activity to less than 10% of that of the wild type protein. These results indicate that genetic variation in the CKM gene may impact tenofovir activation, potentially implicating a risk to patients possessing these genetic variants.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bioengineering of single ncRNA molecule for multi‐targeting against NSCLC
Authors: Petrek Hannah , Ho Pui Yan , Tu Meijuan , Zhang Qianyu , Batra Neelu , Yu Ai‐Ming 
Published: June 2021

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 (ncRNA) in cells that govern target gene expression through mRNA degradation or translational inhibition. Some miRNAs such as miR‐34a, miR‐124 and let‐7c are commonly downregulated in NSCLC tissues/cells and target various oncogenes such as CDK6, STAT3, and Ras. Restoring the expression or function of such dysregulated, tumor suppressive miRNAs represents a new strategy for cancer therapy. Aiming at simultaneously introducing multiple miRNAs in NSCLC cells for multi‐targeting, we employed our recently developed novel ncRNA bioengineering technology to produce a single ncRNA molecule bearing multiple miRNAs, namely combination‐bioengineered RNA agent (CO‐BERA). Our data showed that a variety of target CO‐BERA molecules were successfully expressed in E. coli. We were able to purify recombinant CO‐BERAs to a high degree of homogeneity using anion exchange FPLC methods. We further demonstrated that treatment of human NSCLC cells with CO‐BERAs elevated cellular miR‐34a, miR‐124 and let‐7c levels, reduced protein levels of target genes including CDK6, STAT3 and Ras, and consequently inhibited NSCLC cell proliferation. CO‐BERAs were successful in inhibiting NSCLC patient derived xenograft (PDX) tumor growth in mice. Therefore, using single ncRNA agent for multi‐targeting may be more effective for the treatment of NSCLC.Support or Funding InformationThis study was supported by grants R01GM133888 and U01CA175315 from NIH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anticancer and Chemosensitizing Effects of WEE‐1 Kinase Inhibitor MK‐1775 in Triple Negative (MB‐231) Breast Cancer Cells
Authors: Aviles Nicole Marie Nieves , Mayol Noel , Gonzalez Maria T , Diaz Adriana , Salgado Iris K , Silva Walter , Maldonado Hector 
Published: June 2021

Current breast cancer classification included cells that express estrogen (ER) and progesterone (PR) receptors, those with ERBB2 (HER‐2/Neu) amplification, and those without expression of ER, PR, or amplification of ERBB2 (referred to as triple‐negative or basal‐like breast cancer). Triple negative breast cancers are generally more difficult to treat, are known to be more aggressive and with poor prognosis. Several molecular mechanisms have been identified as potential targets for development of drugs to treat breast cancer including cell cycle checkpoints kinases. The G2/M cell‐cycle protein tyrosine kinase, Wee‐1, plays a crucial role in the checkpoint arrest for DNA repair before mitotic entry. Under normal conditions, cells repair damaged DNA during G1 arrest; however, cancer cells often have a deficient G1/S (p53) checkpoint and depend on a functional G2/M checkpoint for DNA repair. MK‐1775 is a well‐characterized Wee‐1 kinase inhibitor with significant evidence supporting its potential use as anti‐cancer and/or chemosensitizing drug. Working hypothesis proposed for this project was that use of Wee‐1 kinase inhibitors in breast cancer cells can result in profound and significant anticancer and chemosensitizing effects. In order to test this hypothesis, we show previously in a series of dose response experiments using DNA‐Damaging agents or treatments (DNA‐dt: Ex. Cisplatin, 5‐Fluororacil and Doxorubicin) alone and/or in combination with MK‐1775. To continue testing this hypothesis an Immunoblotting bioassay is currently developed for continue monitoring the effects of MK‐1775 in several biochemical markers in triple negative breast cancer cell line. Preliminary results indicated that biochemical marker in triple negative cell line, are time dependent with the different combination of DNA‐dt alone and/or in combination with MK‐1775. Initial preliminary results indicated taken together, our preliminary results are consistent with a potential role of Wee‐1 kinase inhibitors like MK‐17175 as anticancer and/or chemosensitizing agents against triple‐negative or basal‐like breast cancer cells.Support or Funding InformationThe project described was supported by NIGMS/INBRE award P20 GM103475; U.S. Department of Education Title V grant number P031S130068 and Puerto Rico Science Technology and Research Trust. The content is solely the responsibility of the authors and does not necessarily represent the official view of any of the supporting agencies.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antifungal Agents Induce Apoptotic and Non‐Apoptotic Cell Death in Human Melanoma Cells
Authors: Koh David W , Eskender Anna H , Daghistani Ayah T , Jira Lukas R , McKamey Shelby G 
Published: June 2021

Patients with melanoma, the most aggressive form of skin cancer, have poor survival rates due to the resistance of advanced stage tumors to chemotherapy. This resistance appears to involve the dysregulation of apoptosis, such that inhibition of apoptotic progression allows melanoma cells to survive after treatment. We previously reported a potentially novel treatment for melanoma, where antifungal agents led to profound decreases in growth and proliferation of human melanoma cells. Our objective here was to analyze cell death pathways induced in human melanoma cell lines following treatment with antifungal agents. Cell death pathways were investigated via flow cytometry, cell proliferation assays, and immunoblotting after treatments.Several lines of human melanoma and normal skin cells were analyzed. Apoptosis, via flow cytometry and analysis of activated caspases and PARP1 cleavage, was observed in all melanoma lines after treatment with clotrimazole and econazole. Caspase‐independent cell death was also observed in most melanoma lines after similar treatments. No extensive cell death was induced by these treatments in noncancerous human skin cells. These results indicated that antifungal agents selectively induced cytotoxicity in human melanoma lines. Because varying levels of cell death were observed among different melanoma lines after antifungal treatment, further studies will be required to ascertain the reason for these differential responses to treatment. Taken together, these results indicated that clotrimazole and econazole initiated apoptotic and non‐apoptotic cell death in human melanoma cells.In conclusion, this study demonstrated the induction of apoptotic and non‐apoptotic cell death in human melanoma skin cell lines after treatment with antifungal agents. Differential levels of various cell death pathways were observed among all melanoma lines. This was corroborated by the various levels of efficacious treatment produced by these agents. Taken together, we conclude that clotrimazole and econazole induce cell death in human melanoma cells. These agents thus appear to have in vitro efficacy toward the treatment of human melanoma.Support or Funding InformationThis research was supported in part by the Bower, Bennet & Bennet Endowed Chair Award from the Ohio Northern University College of Pharmacy and the Ohio Northern University Summer Research stipend.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Constraining Multi‐Drug Resistance in Breast Cancer Cells by Energy Restriction
Authors: Omar Hany A. , Zaher Dana M. , Tolba Mai F. , Al‐Tel Taleb H. 
Published: June 2021

The resistance of breast cancer to chemotherapy constitutes a major roadblock for a successful treatment. The development of multidrug resistance (MDR) is one of the common mechanisms for resistance to various chemotherapeutic agents including doxorubicin (DOX). In this context, we tested the hypothesis that energy restriction mimetic agents (ERMAs) such as OSU‐GC5 could synergize the antitumor effects of DOX through restraining the availability of energy that is essential for the activity of MDR efflux carriers. Subclones of MCF‐7 and MDA‐MB231 breast cancer cell lines resistant to DOX have been established by subjecting the cells to increasing concentrations of DOX over 6 months. The MDR phenotype was verified via the upregulated expression of ATP binding cassette (ABC) protein ABCC1, ABCB1 and ABCG2 together with a reduced cellular accumulation of DOX. Fluorescence‐activated cell sorting analysis indicated that the percentage of CD44+/CD24− stem cell‐like population was enriched in the resistant subclones. Combination analysis demonstrated a synergistic antitumor interaction between OSU‐CG5 and DOX accompanied by augmented activation of mitochondrial apoptosis and AMPK signaling as verified by Western blot analysis. Co‐treatment with OSU‐CG5 and DOX significantly reduced cellular ATP content and NADP+/NADPH ratio, while it significantly enhanced ROS production. Concomitant exposure to ROS scavenger, N‐acetylcysteine, eliminated the synergy between OSU‐CG5 and DOX. This study recommended OSU‐CG5 as a promising adjuvant to overcome MDR resistance. This combinatorial strategy can be extrapolated to breast cancer treatment regardless of the hormone receptor expression status, which encourages future research in syngeneic murine tumor xenograft models.Support or Funding InformationThis work was supported by a research grant from Al Jalila Foundation, United Arab Emirates (Grant number AJF201610)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A PF‐3845‐induced novel lipid fraction in mouse medullary interstitial cells causes diuresis and natriuresis via a CB1‐mediated pathway
Authors: Daneva Zdravka P , Dempsey Sara , Ahmad Ashfaq , Ritter Joseph 
Published: June 2021

The relationship between the endocannabinoid system in the renal medulla and the long‐term regulation of blood pressure is not understood. To investigate the possible role of the endocannabinoid system in renomedullary interstitial cells, mouse medullary interstitial cells (MMICs) were obtained, cultured and characterized for their responses to treatment with a selective inhibitor of fatty acid amide hydrolase (FAAH), PF‐3845. Treatment of MMICs with PF‐3845 increased cytoplasmic lipid granules detected by Sudan Black B staining and multilamellar bodies identified by transmission electron microscopy. HPLC analyses of lipid extracts of MMIC culture medium revealed a 205nm‐absorbing peak that showed responsiveness to PF‐3845 treatment. The biologic activities of the PF‐3845‐induced product (PIP) isolated by HPLC were investigated in anesthetized, normotensive surgically‐instrumented mice. Intravenous and intramedullary infusions of PIP at low doses resulted in a vasodepressor, diuretic and natriuretic effects. Pharmacological and genetic ablation of cannabinoid 1 receptors resulted in the loss of the vasodepressor, diuretic and natriuretic effect of PIP. Radioactive binding assays showed that PIP competes for binding to CB1 receptors in the presence of a full agonist (CP 55,940). PIP did not elevate urine and sodium excretion in a genetic knockout mouse model of FAAH. A PIP‐induced decrease in blood pressure at high doses of PIP was similar between FAAH WT and FAAH KO mice. These data support a model whereby PF‐3845 treatment of MMICs results in increased secretion of a neutral lipid which acts directly to promote diuresis and natriuresis via a CB1‐mediated mechanism. The diuretic and natriuretic responses to PIP are eliminated by inhibition of FAAH, suggesting an undergoing sensitization to PIP in FAAH KO mice. Efforts to identify the structure of the PF‐3845‐induced lipid and its relationship to the previously proposed renomedullary antihypertensive lipids are ongoing.Support or Funding InformationThis work was supported by the National Institute of Diabetes and Digestive and Kidney Disorders grant [grant R01DK102539] and the National Institute on Drug Abuse [grant P30DA033934]. S.D. was supported by the National Institute on Drug Abuse [training grant T32DA007027].This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Luteolin attenuates glycerol‐induced acute renal failure through modulation of Kim‐1/NF‐κB /Nrf2 signaling pathways
Authors: Oyagbemi Ademola Adetokunbo , Omobowale Temidayo Olutayo , Ola‐Davies Olufunke Eunice , Asenuga Ebunoluwa Racheal , Ajibade Temitayo Olabisi , Adejumobi Olumuyiwa Abiola , Afolabi Jeremiah Moyinoluwa , Ogunpolu Blessing Seun , Falayi Olufunke Olubunmi , Hassan Fasilat Oluwakemi , Nabofa Williams Enivwenaye , Saba Adebowale Benard , Adedapo Adeolu Alex , Yakubu Momoh Audu 
Published: June 2021

Acute renal failure (ARF) has been documented as a life‐threating disease with high morbidity and mortality. We investigated the protective effect of Luteolin against ARF. In the study, forty‐male Wistar albino rats were randomly divided into four groups (n = 10). Group A received normal saline. Group B received glycerol (10 ml/kg BW, 50% v/v in sterile saline, i.m.). Groups C and D were pre‐treated with Luteolin 100 and 200 mg/k for 7 days, and thereafter administered Glycerol (10 ml/kg BW, 50% v/v in sterile saline, i.m.). Administration of glycerol significantly increased blood pressure, renal oxidative stress, serum creatinine and reduced nitric oxide (NO) bioavailability. Glycerol administration enhanced expressions of kidney injury molecule 1 (Kim‐1), nuclear factor kappa beta (NF‐κB) and nuclear factor‐erythroid‐2‐related factor 2 (Nrf2). Luteolin pre‐treatment normalized blood pressure, reduced markers of oxidative stress, renal damage, and improved NO bioavailability. Together, Luteolin attenuates glycerol‐induced acute renal failure through modulation of Kim‐1/NF‐κB /Nrf2 signaling pathways. Hence, Luteolin might be a novel therapeutic target for the treatment of acute renal failure.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lysosome Dysfunction and Medial Calcification in the Arterial Wall of Smooth Muscle Cell‐Specific Smpd1 Transgenic Mice: A Ceramide‐Mediated Vasculopathy
Authors: Bhat Owais M , Yuan Xinxu , Lohner Hannah , Li Pin‐Lan 
Published: June 2021

It has been reported that multivesicular body (MVB) trafficking and release of exosomes are a hallmark of vascular calcification. Since ceramide is a crucial regulator of lysosome function, the present study was performed to test whether lysosomal acid sphingomyelinase (its mouse gene code is Smpd1)‐derived ceramide contributes to the exosome secretion from smooth muscle cells (SMCs) and consequently lead to arterial calcification. In Smpd1trg/SMcre mice with SMC‐specific overexpression of Smpd1 gene, a high dose of Vit D (500,000 IU/kg/day) induced much more aortic and coronary arterial medial calcification compared to their littermates (Smpd1fl/fl/WT and WT/WT mice), as detected by Alizarin Red S and Von Kossa staining. It was also found that there was more significantly increased expression of RUNX2 and osteopontin in the coronary and aortic media of Smpd1trg/SMcre mice than their littermates, indicating a SMC phenotypic transition to osteogenic in these SMC‐specific Smpd1 transgenic mice. When these mice received treatment with amitriptyline, an Asm inhibitor, the calcification and phenotypic switch in both coronary and aortic medial SMCs were reversed. Furthermore, Smpd1trg/SMcre mice were found to have increased annexin 2 (AnX2) and alkaline phosphatase (ALP) levels (exosome markers) in the arterial wall, which was accompanied by reduced co‐localization of lysosome marker (LAMP‐1) with MVB marker (VPS16), a parameter for lysosome‐MVB interaction. All these changes related to lysosome function and exosome release from SMCs in Smpd1trg/SMcre mice were substantially attenuated by amitriptyline treatment. These data indicate that lysosomal ceramide plays a critical role in phenotype change and exosome release in SMCs, which may contribute to the control of mineral metabolism or deposition in the arterial wall.Support or Funding InformationSupported by NIH grants HL057244, HL075316 and DK120491This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of α5β1integrin with the clinically validated small peptide, ATN‐161 stabilizes cerebral vasculature, reduces inflammation, and decreases blood‐brain barrier permeability after experimental ischemic stroke
Authors: Edwards Danielle , Salmeron Katie , Lukins Douglas , Trout Amanda , Fraser Justin , Bix Gregory 
Published: June 2021

Stroke is the second leading cause of death and the leading cause of long‐term disability worldwide. Blood‐brain barrier (BBB) dysfunction exacerbates reperfusion‐induced injury after recanalization in ischemic stroke. Endothelial cell integrin receptors, specifically the β1 subtype, play a direct role in this BBB dysfunction through regulation of barrier‐forming tight junction (TJ) proteins. We hypothesize that inhibition of a specific β1 integrin subtype, α5β1, will stabilize the BBB, and thereby reduce infarct volumes and improve functional recovery after experimental stroke.MethodMale C57BLJ6 mice underwent transient middle cerebral artery occlusion for 1 hour. Upon reperfusion, vehicle or the clinically validated α5β1 inhibitor, ATN‐161 (1mg/kg), was injected intraperitoneally and repeated 24 and 48 hours later. Infarct volume (TTC and MRI – DTI sequence) and edema volume (T2‐weighted MRI) was determined on PSD3. Functional recovery was determined by an 11‐point Neuroscore on PSD1‐PSD7. Physiological measurements, including pulse distention, heart rate and body temperature, were obtained before, during and after the initial dose of ATN‐161. Immunohistochemical analysis of α5β1, claudin‐5, CD45 (pan‐leukocyte), and IgG, expression was performed on PSD3. qPCR analysis on claudin‐5, collagen IV, MMP‐9, IL‐1β, and CXCL12 were performed on PSD3.ResultsATN‐161 administration reduced infarct volume, edema volume, and functional deficit. ATN‐161 also resulted in cerebral vascular stabilization by a decrease in MMP‐9 and increases in claudin‐5, and collagen IV. Furthermore, IL‐1β and CD45 was reducedupon ATN‐161 administration, suggesting a decrease in the post‐stroke inflammatory response. Together, the cerebrovascular stabilization and decrease in inflammation resulted in less IgG extraversion, suggesting a more intact BBB. Collectively, administration of ATN‐161 post‐stroke produced significant benefits, and thus could represent a novel stroke therapy worthy of further investigation.Support or Funding InformationThis work was supported by NIH R01NS065842.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.