bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evaluation of Polyamine Transport Inhibitors in a Drosophila Epithelial Model
Authors: von Kalm Laurence , Wang Minpei , Phanstiel Otto 
Published: June 2021

Increased polyamine biosynthesis activity and an active polyamine transport system are characteristics of many cancer cell lines and polyamine depletion has been shown to be a viable anticancer strategy. Polyamine levels can be depleted by difluoromethylornithine (DFMO), however, malignant cells frequently circumvent DFMO therapy by up‐regulating polyamine import. Therefore, there is a need to develop compounds that inhibit polyamine transport. We have previously shown that the pattern of uptake of a series of polyamine analogues in a Drosophila model epithelium shares many characteristics with mammalian cells, indicating a high degree of similarity between the mammalian and Drosophila polyamine transport systems. Here, we focused on the utility of the Drosophila epithelial model to identify and characterize polyamine transport inhibitors. We show that a previously identified inhibitor of transport in mammalian cells has a similar activity profile in Drosophila. The Drosophila model was also used to evaluate two additional transport inhibitors. We further demonstrate that a cocktail of polyamine transport inhibitors is more effective than individual inhibitors, suggesting the existence of multiple transport systems in Drosophila. Our findings reinforce the similarity between the Drosophila and mammalian transport systems and the value of the Drosophila model to provide inexpensive early screening of molecules targeting the polyamine transport system.Support or Funding InformationUniversity of Central Florida, College of Sciences Seed AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Knockout of Fatty Acyl‐CoA Synthetase ACSVL3 in Glioma Cells Produces Diverse Metabolic Alterations in Non‐Lipid Pathways
Authors: Watkins Paul , Shi Xiaohai , Kolar Elizabeth , Clay Emily , Liu Yanqiu 
Published: June 2021

Knockout (KO) of the fatty acid‐activation enzyme very long chain acyl‐CoA synthetase 3 (ACSVL3; SLC27A3) in glioblastoma cells reduced their malignant growth properties both in vitro and in xenografts. KO U87 glioma cells grew at a slower rate, became adherence‐dependent, and were less invasive than parental U87 cells. KO cells produced fewer, slower‐growing subcutaneous and intracranial tumors when implanted in NOD‐SCID mice. To understand the mechanisms underlying these changes, we investigated several possibilities. We first hypothesized that lack of ACSVL3 might reduce the rate of incorporation of labeled fatty acids into membrane phospholipids; however, no differences were observed. We then investigated the possibility that lack of ACSVL3 affected either apoptosis or autophagy, but detected no differences in these processes between control and KO U87 cells. We next examined cell cycle, and found that KO produced significant changes. In non‐synchronized cells, parental U87 cells were mainly in G0/G1, while ACSVL3 KO cells were mainly in S‐phase. After synchronization by serum starvation, both U87 and KO cells started out mainly in G0/G1, but by 12 hours ACSVL3 KO cells shifted significantly toward S‐phase. Consistent with this finding, the levels of several cyclins and cyclin‐dependent kinases were found to be altered in KO cells. In addition to effects on lipid metabolism, proteomic and metabolomics profiling indicated that ACSVL3 KO produced changes in glucose and energy metabolism. Even though protein levels of glucose transporters GLUT1 and GLUT3 were reduced by KO, cellular uptake of labeled 2‐deoxyglucose was unaffected. Glucose oxidation to CO2 was reduced 40% by ACSVL3 depletion, and the cellular glucose level was 25% higher in KO cells. Glycolytic enzymes were upregulated by KO, but metabolic intermediates were essentially unchanged. Surprisingly, lactate production and the levels of both LDHA and LDHB were elevated by ACSVL3 KO. The activity of the pentose phosphate pathway was found to be lower in KO cells. Citric acid cycle enzymes, electron transport chain complexes, and ATP synthase protein levels were all reduced by ACSVL3 depletion. Mitochondria were elongated in KO cells, but had a more punctate morphology in U87 cells. We conclude that beneficial effects of ACSVL3 depletion in human glioblastoma cells are the result of alterations in diverse metabolic processes that are not directly related to fatty acid and/or lipid metabolism.Support or Funding InformationSupported by Institutional Funds.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthesis and characterization of BODIPY‐FL‐cyclosporine A as a substrate for both human and mouse multidrug resistance‐linked P‐glycoprotein
Authors: Sajid Andaleeb , Natarajan Raju , Lusvarghi Sabrina , Vahedi Shahrooz , Swenson Rolf , Ambudkar Suresh V 
Published: June 2021

Generation of fluorescent conjugates of small molecules and existing drugs has facilitated their use in addressing cellular functions and pharmacological analysis. These compounds interact with several proteins as substrates or inhibitors, thus allowing the development of unique fluorescence‐based methods to study in vivo localization and the molecular mechanisms. Pglycoprotein (P‐gp) is an ATP‐binding cassette transporter that effluxes most anti‐cancer drugs from cells, contributing to the development of drug resistance in cancer cells. P‐gp, which exports toxic metabolites and xenobiotics, is expressed on the surface of epithelial cells of the intestine, kidney, liver, placenta, adrenal gland and endothelial cells at blood‐brain barrier. With the aim of developing an effective probe to study drug‐transport by P‐gp in various model systems including cultured cells, mice and zebra fish, we synthesized a BODIPY‐FL conjugate of cyclosporine A (BD‐CsA). After synthesis and characterization of its chemical purity, BD‐CsA was compared with the currently available 7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl (NBD)‐CsA probe in terms of its usage as a substrate of human and mouse P‐gp. Using a BacMam baculovirus HeLa cell expression system and a flow cytometry‐based transport assay, we found that BD‐CsA is recognized as a substrate by both human and mouse P‐gp. The rate of efflux of BD‐CsA by human P‐gp is similar to that of NBD‐CsA (T1/2 of 5.0 min and 3.0 min for BD‐CsA and NBD‐CsA, respectively). In flow cytometry assays, the fluorescence intensity of BD‐CsA was almost 10‐times higher than that of NBD‐CsA due to the high fluorescence quantum yield of the BODIPY fluorophore, allowing us to use significantly lower concentrations of BD‐CsA to achieve the same fluorescence levels. The transport of BD‐CsA was inhibited by the P‐gp inhibitor tariquidar, with similar IC50 values as for NBD‐CsA transport (21.6 nM for BD‐CsA and 25.8 nM for NBD‐CsA). We also observed that both BD‐CsA and NBD‐CsA partially inhibited the ATPase activity of P‐gp. In silico docking of BD‐CsA and NBD‐CsA to the homology model of human P‐gp indicates that both probes bind in the drug‐binding pocket of Pgp with similar docking scores. Thus, we demonstrate that BD‐CsA is a sensitive and efficient fluorescent substrate of P‐gp that can be used to study the transporter's function in both in vitro and in vivo settings.Support or Funding InformationThis research was funded by the Intramural Research Program of the National Institutes of Health, the National Cancer Institute, Center for Cancer ResearchThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Function of the KCNQ1 Potassium Ion Channel in the Human Heart
Authors: Link Tina , Jacobson Reagan , Lee Chase , Maraghi Sophie , Mohan Siddharth , Shabazaz Tara , Vemana Riya , Wright Aryn 
Published: June 2021

KCNQ1 is a homotetrameric voltage‐gated potassium ion channel subunit protein. KCNQ1 is the alpha‐subunit of the ion channel and forms heteromultimers with protein members of the KCNE family (“beta subunits”) to create a pore in the membrane of a cell. Two of these beta subunits are KCNE1 and KCNE3. Under physiological conditions, four KCNQ1 alpha subunits combine with two or four beta subunits to form the potassium ion channel. The alpha subunits form the structure of the channel whereas the beta subunit binds onto the channel and regulates its activity. KCNQ1 is embedded in the membranes of numerous types of cells, including cardiac myocytes, epithelial cells, and cells in the human inner ear. In human cardiac myocytes, KCNQ1 and KCNE1 work to regulate the rhythm of the heart by controlling the repolarization phase of the cardiac action potential. Almost three hundred mutations have been identified in the KCNQ1 gene, which is located on chromosome 11. The majority of these are missense mutations and alter the protein's structure to the extent that the flow of potassium ions out of the cardiac cells is disrupted. The most common condition resulting from a mutation in KCNQ1 in hereditary Long QT Syndrome (LQTS), which is characterized by a delay in the ventricular repolarization component of a heart beat. Symptoms associated with LQTS include dizziness, fainting, and potentially seizures and sudden death. In rare cases, a mutation in KCNQ1 can also cause further complications, which include atrial fibrillation, Short QT syndrome, and Jervell and Romano‐Ward syndrome. Treatments are available for LQTS, such as making lifestyle changes, taking beta blockers, and implanting a defibrillator. Current research in medication for LQTS is centered around compounds such as hexachlorophene that can target the individual subunits in the ion channel as opposed to the entire channel. Pairing compounds with this ability may provide enough correction to the subunits that LQTS can be effectively “cured.” The Walton High School SMART Team has designed a 3D model of the KCNQ1 ion channel using JMOL to investigate the relationship between structure and function.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Podocyte‐Specific Rescuing and Deletion of Acid Ceramidase Gene in Mouse Glomeruli
Authors: Zhang Qinghua , Li Guangbi , Yuan Xinxu , Bhat Owais M , Lohner Hannah , Li Pin‐Lan 
Published: June 2021

We have recently developed a podocyte‐restricted acid ceramidase (AC) gene knockout mouse model (Asah1fl/fl/Podocre, Asah1 is a code of mouse AC gene), which was shown to produce podocyte dysfunction and consequent minimal change disease (MCD). The present study attempted to develop somatic gene intervention methods to perform podocyte‐specific gene deletion, rescuing and silencing of this AC gene in Asah1fl/fl/Podocre mice and their control littermates including Asah1fl/fl/WT mice, which could exclude the potential development or compensatory influence given that Asah1 gene was embryonically deleted in podocytes in those animal strains. We first constructed 2 different vectors which can be transfected to change AC gene expression including a podocyte‐specific AC gene (podo‐Asah1) expression vector and a podocyte‐specific Cre gene (podo‐Cre) expression vector. The pEZX vector containing podocin promoter was used to generate Asah1 and Cre full length gene expression plasmids, which were transfected into Asah1fl/fl/Podocre mice to rescue Asah1 gene and into Asah1fl/fl/WT mice to delete floxed Asah1 gene, respectively. Using isolated podocytes from 2 different mouse strains, we confirmed by immunofluorescence staining, real‐time PCR and Western blot analysis that these vectors could be successfully transfected into podocytes and produced corresponding effects to alter Asah1 gene expression, for example, rescuing of this gene in Asah1fl/fl/Podocre podocytes and deletion of this gene in Asah1fl/fl/WT podocytes. In animal experiments, we used microbubble ultrasound transfection techniques to introduce different plasmids into the kidneys. It was found that Asah1 gene was rescued in podocytes of Asah1fl/fl/Podocre mice by podo‐Asah1 plasmids and deleted in Asah1fl/fl/WT mice by podo‐Cre plasmids. Functionally, we demonstrated that increased Asah1 expression in podocytes decreased desmin levels, but enhanced podocin expression, indicating restoration of podocyte function. It also enhanced the interaction of multivesicular bodies (MVBs) and lysosomes as shown by increase in Vps16 and Lamp‐1 colocalization, which resulted in decreased exosome release from podocytes. Taken together, our results suggest that somatic transfection of podocin promoter‐driven Asah1 and Cre gene expression vectors produced podocyte‐restricted gene changes in different gene engineered animal models for AC function studies.Support or Funding Informationsupported by NIH grant DK54927, DK120491 and HL057244This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acid Sphingomyelinase Deficiency Protects Mitochondria and Improves Function Recovery after Traumatic Brain Injury
Authors: Novgorodov Sergei A. , Voltin Joshua R. , Riley Christopher L. , Gudz Tatyana I. 
Published: June 2021

Traumatic brain injury (TBI) is one of the leading causes of disability worldwide and a prominent risk factor for neurodegenerative diseases. The expansion of nervous tissue damage after the initial trauma involves a multifactorial cascade of events including excitotoxicity, oxidative stress, inflammation and deregulation of sphingolipid metabolism that further mitochondrial dysfunction and secondary brain damage. Our studies establish a fundamental role of acid sphingomyelinase (ASM) in the multi‐faceted mechanisms governing secondary brain injury after TBI. The data indicate that TBI‐triggered activation of ASM and upregulation of mitochondrial sphingosine resulted in mitochondrial respiratory chain malfunction leading to an enhanced assembly and activity of the NLRP3 inflammasome, a pivotal factor promoting the neuroinflammatory response to brain injury. The results of our studies provide further support for the emerging role of ASM in repressing the cell maintenance and quality control mechanism, autophagy, which is essential for the neural cell survival after the injury. Thus, knocking down ASM rescued mitochondrial defect, augmented the autophagic flux, downregulated the neuroinflammation and improved the brain function recovery after TBI. Importantly, ASM is involved in the initial stages of progression of astrogliosis, a brain defense mechanism wherein activated astrocytes minimize spread of the neuroinflammation and further tissue repair and regeneration in response to TBI. Our novel findings reveal that ASM, a critical player promoting secondary brain injury and neuroinflammation is uniquely positioned to be a promising target for the development of effective pharmacological treatment of patients with TBI.Support or Funding InformationSupported by NIH grant R01NS083544 (TIG) and VA Merit Award I01BX002991 (TIG).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ULK1‐dependent Mitophagy Is Essential for Maintaining Cardiac Function during High Fat Diet‐induced Diabetic Cardiomyopathy
Authors: Tong Mingming , Saito Toshiro , Zhai Peiyong , Oka Shinichi , Sadoshima Junichi 
Published: June 2021

Diabetic patients develop cardiomyopathy characterized by hypertrophy, diastolic dysfunction, lipotoxicity, and mitochondrial dysfunction. How mitochondrial function is regulated in diabetic cardiomyopathy remains poorly understood. Mice were fed either a normal diet (ND) or a high fat diet (HFD, 60 kcal % fat). Mitophagy, evaluated with Mito‐Keima, was increased after 3 weeks of HFD feeding (mitophagy area: 8.3% per cell with ND and 12.4% with HFD) and continued to increase after 20 weeks (p<0.05). Although we have shown recently that mitophagy during the early phase of HFD feeding is mediated by Atg7‐dependent mechanisms, the mechanisms mediating mitophagy in the heart during the chronic phase of HFD feeding remain poorly understood. Phosphorylation of ULK1 was activated and Rab9 protein level was increased in the mitochondrial fraction within 20 weeks of HFD consumption (p<0.05). By isolating adult cardiomyocytes from GFP‐Rab9 transgenic mice fed HFD, we discovered that mitochondria were sequestrated by Rab9‐positive ring‐like structures. Since ULK1 regulates Rab9‐dependent mitophagy, we fed ULK1 cKO mice with HFD for 20 weeks. In wild type (WT) mice, cardiac hypertrophy and diastolic dysfunction (EDPVR = 0.051±0.009 in ND and 0.115±0.006 in HFD) were induced after 20 weeks of HFD feeding (p<0.05). By crossing Tg‐Mito‐Keima mice with ULK1 cKO mice, we found that downregulation of ULK1 impaired mitophagy in response to ND or 20 weeks of HFD consumption (p<0.05). Deletion of ULK1 exacerbated diastolic dysfunction (EDPVR=0.115±0.006 in WT and 0.162±0.021 in ULK1 cKO, p<0.05) and even induced systolic dysfunction (ESPVR=22.74±2.13 in WT and 16.78±2.12 in ULK1 cKO, p<0.05) during HFD feeding. Electron microscopic analyses indicated that the mitochondrial cristae structure was disrupted more severely in ULK1 cKO mice with HFD feeding than control mice (p<0.05). In summary, genetic disruption of ULK1‐Rab9‐dependent mitophagy during the chronic phase of HFD feeding exacerbates mitochondrial dysfunction, thereby facilitating the development of diabetic cardiomyopathy. ULK1‐Rab9‐dependent mitophagy serves as an essential quality control mechanism for cardiac mitochondria during HFD feeding.Support or Funding InformationThe project was supported by AHA and NIH (5R01HL138720‐02).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mean Platelet Volume in Patients with Regulated and Unregulated Type 2 Diabetes Mellitus According to Sex
Authors: Kama Tabitha Supriya , Nayakanti Abraham Ratna J , CS Jaya Prakash , Srinivasan KR , Koteswary R 
Published: June 2021

Type 2 diabetes mellitus(DM) is a metabolic disorder with a prevalence of 8.8% in adults in India and 425 million cases worldwide. The high prevalence and morbidity make it a worldwide health problem. Type 2 DM is a part of metabolic syndrome which comprises of hypertension, dyslipidemia, increased procoagulation factors and impaired fibrinolysis. HbA1c is a Glycated hemoglobin is a form of hemoglobin that is measured primarily to identify the three‐month average plasma glucose concentration in diabetics and is measured by automated ion‐exchange high performance liquid chromatography (Bio‐Rad Variant II, Hercules, CA). Hyperglycemia in diabetic patients is thought to have a harmful effect on the blood vessels. Platelet volume is a marker of platelet function and activation. It can be quantified as mean platelet volume (MPV) by clinical hematology analyzers. Platelets are involved in homeostatic process and have an important role in atherosclerosis and arterial thrombosis. When vascu‐lar injury occurs, platelets adhere to dam‐aged endothelium to form platelet plug. It has been reported that plate‐lets from diabetic patients synthesize more thromboxane than normal platelets. To compare the MPV between regulated (HbA1c<6.5%) and nonregulated (HbA1c > 6.5%) diabetics.MATERIALS AND METHODSStudy design‐ Cross sectional study, Study duration‐ 06 months; February 2018–July 2018. Sample size‐ Based on the article the proportion used to measure the parameter MPV is 8.8% with an alpha of 5 % the corresponding z value is ‐1.95996398454005. Using the below formula with the minimum percentage difference to be deemed clinically significant as 4.7%. The study outlines the importance of MPV in regulated and non regulated type 2 DM.Support or Funding InformationASIPThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SIRT3: a potential target for CHF?
Authors: Liu Peiqing 
Published: June 2021

Pathological cardiac hypertrophy is a maladaptive response in a variety of organic heart disease (OHD), which is characterized by mitochondrial dysfunction that results from disturbed energy metabolism. SIRT3, a mitochondria‐localized sirtuin, regulates global mitochondrial lysine acetylation and preserves mitochondrial function. However, the mechanisms by which SIRT3 regulates cardiac hypertrophy remains to be further elucidated. In this study, we firstly demonstrated that expression of SIRT3 was decreased in Angiotension II (Ang II)‐treated cardiomyocytes and in hearts of Ang II‐induced cardiac hypertrophic mice. In addition, SIRT3 overexpression protected myocytes from hypertrophy, whereas SIRT3 silencing exacerbated Ang II‐induced cardiomyocyte hypertrophy. In particular, SIRT3‐KO mice exhibited significant cardiac hypertrophy. Mechanistically, we identified NMNAT3 (nicotinamide mononucleotide adenylyltransferase 3), the rate‐limiting enzyme for mitochondrial NAD biosynthesis, as a new target and binding partner of SIRT3. Specifically, SIRT3 physically interacts with and deacetylates NMNAT3, thereby enhancing the enzyme activity of NMNAT3 and contributing to SIRT3‐mediated anti‐hypertrophic effects. Moreover, NMNAT3 regulates the activity of SIRT3 via synthesis of mitochondria NAD. Taken together, these findings provide mechanistic insights into the negative regulatory role of SIRT3 in cardiac hypertrophy. Sirtuin 3 (SIRT3), a mitochondrial deacetylase that may play an important role in regulating cardiac function and a potential target for CHF.Support or Funding InformationThis work was supported by grants from the National Natural Science Foundation of China (No 81673433, No 81026548 and No 81273499), Team item of the Natural Science Foundation of Guangdong Province (NoS2011030003190)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
miR‐141 inhibition protects cardiomyocytes from anoxia/reoxygenation‐induced mitochondrial dysfunction
Authors: Zhang Hao , Yan Jun , Wang Hao , Tao Aibin , Rui Tao 
Published: June 2021

BackgroundMitochondrial dysfunction plays an important role in ischemia/reperfusion (I/R) ‐induced myocardial injury. MicroRNAs are involved in myocardial injury after I/R. It has been reported that miR‐141 contributes to mitochondrial dysfunction in mice with diabetes. The aim of the present study is to dertmine whether the miR‐141 contributes to I/R‐induced myocardial injury and the underlying mechanism.MethodsHL‐1 cells were challenged with an anoxia/reoxygenation (A/R). miR‐141 expression in HL‐1 cells was detected by qRT‐PCR. Mitofusin‐2 (Mfn2) was assessed with Western blot. HL‐1 cellular oxygen consumption rate (OCR) was assessed with Seahorse XF24 analyzer.ResultsA/R challenge to HL‐1 cells increased miR‐141 expression and peaked at 12 hours after the reoxygenation. A/R challenge resulted in decreased mitochondrial membrane potential, OCR and ATP production of the cardiomyocytes, as well as increased LDH activity in the supernatant of the cardiomyocytes. The above changes were attenuated by transfection of the cardiomyocytes with miR‐141 inhibitor before the A/R. Overexpression of miR‐141 in the HL‐1 cell exaggerated the A/R‐induced mitochondrial dysfunction and cardiomyocyte injury. The A/R challenge to HL‐1 cells led to a decreased Mfn2 expression. Transfection of HL‐1 cells with miR‐141 mimics suppressed Mfn2 expression.ConclusionOur data indicate that miR‐141 is involved in A/R‐induced mitochondrial dysfunction by modulating Mfn2 protein expression.Support or Funding InformationThe study was supported by Natural Science Foundation of Jiangsu Province (BK20151332) and Natural Science Foundation of China (81370333).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.