bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ibuprofen cases Proteasome dysfunction in mice liver
Authors: Gomes Aldrin , Tiwari Shuchita , Sule Rasheed 
Published: June 2021

Introduction and objectivesPrevious clinical studies have suggested that nonsteroidal anti‐inflammatory drugs (NSAIDs) are associated with increased risk of stroke and cardiovascular disease. Acetaminophen, the active ingredient in Tylenol, is not an NSAID and is associated with increased risk of liver toxicity. The common use of NSAIDs for pain and fever relief is a concern but it is unclear if NSAIDs is also causing adverse effects on other tissues such as the liver, kidney and brain. Previous research in our laboratory has suggested that NSAIDs cause proteasome dysfunction in cardiac tissue. To investigate the possibility that NSAIDs also influence liver function, ibuprofen treated mice were used to determine if this NSAID affects the ubiquitin proteasome system (UPS). The UPS is critical for cell function as it is responsible for degradation of 60–80% of intracellular proteins.Method/ResultsEight‐week‐old male and female C57BL/6J mice were randomly assigned to one of 4 groups: (A) control (B) Group treated with ibuprofen (C) Group treated with ibuprofen + vitamin C (D) Group treated with vitamin C alone. Mice were treated with water or ibuprofen (100 mg/kg) and vitamin C (150 mg/kg) dissolved in drinking water for 6 weeks. Liver from 6 week treated animals were homogenized and cytosol fractions obtained. The three independent proteolytic proteasome activities, β1 caspase‐like activity, β2 trypsin‐like activity, and β5 chymotrypsin‐like activity were measured in the different cytosolic liver fractions. The proteasome inhibitor bortezomib was utilized to ensure the activity measured was due to the proteasome.Semi‐quantitative western blotting suggests that the 20S proteasome amount was similar in all 4 groups but the immunoproteasome subunit β5i was increased in both male and female ibuprofen treated and ibuprofen with vitamin C groups. The β1 and β2 proteasome activities were significantly decreased in both male and female mice treated with ibuprofen. Although inclusion of vitamin C with the ibuprofen did not fully restore proteasome activity, it resulted in an improvement in the β1 proteasome activity compared to ibuprofen treated mice. The β5 proteasome activity was also significantly decreased in both male and female mice treated with ibuprofen. However, this decrease was significantly more pronounced in female mice than in male mice suggesting gender‐specific effects of ibuprofen on proteasome function.ConclusionThese results suggest that ibuprofen significantly disrupts the UPS and causes proteasome dysfunction in liver tissue. The experimental data also suggests that proteasome dysfunction in livers from ibuprofen treated mice differs in female and male mice.Support or Funding InformationAmerican Heart Association Award #16GRNT31350040This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High Throughput Phenotypic Analysis of Circulating Tumor Cells from Pancreatic Adenocarcinoma Patients via Dielectrophoresis and Image Flow Cytometry
Authors: Marra Andre Antonio , Kim Dong , Carpenter Chloe , Varadhachary Gauri , Katz Mathew , Greyscone Peter , Alvarez Hector , Maitra Anirban 
Published: June 2021

Effective management of pancreatic ductal adenocarcinoma (PDAC) has remained elusive1. Even with large scale sequencing efforts of cancer genomes providing potential for guidance in informing tumor characterization and therapeutic strategies, the inaccessibility of primary and metastatic tissue precludes the opportunity for further workup following initial tumor sampling2. To circumvent this issue, we utilize circulating tumor cells (CTCs), or those cells shed by primary and metastatic tissue, to noninvasively monitor tumor evolution and changing patterns of drug susceptibility3. As current CTC enrichment methodologies typically involve the use of antibody (anti‐EPCAM) based capture approaches, the ability to detect phenotypic changes as a result of adaptive gene expression patterns such as cells undergoing epithelial to mesenchymal transition (EMT) is lost. We thus aim to isolate CTCs using an antigen independent platform based on a cell's dielectrophoretic (DEP) properties which allow characterization of multiple subpopulations of cells which may have clinical relevance.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ULK2 Regulates p62‐ and NBR1‐Dependent Selective Autophagy In Skeletal Muscle
Authors: Fuqua Jordan , Mere Caleb , Bloome Jay , Melo Vitor , Scudese Estevao , Bae Dam , Turner Kristen , Kronemberger Ana , Lira Vitor 
Published: June 2021

Autophagy is an anciently conserved pathway responsible for the degradation of long‐lived proteins, protein aggregates, and organelles, thereby contributing to efficient protein homeostasis. Autophagy is stimulated by nutrient deprivation and is required for certain beneficial adaptations of exercise. Conversely, insufficient autophagy is a common feature of muscle diseases, obesity, type 2 diabetes, and aging; yet the regulation of autophagy is incompletely understood at the molecular level. Here, our goal was to define the role of muscle unc‐51 like autophagy activating kinase 2 (ULK2), and contrast with that of its close homolog ULK1, in regulation of autophagy. To do so, we performed 2 complementary studies. First, DNA plasmids encoding either Ulk1 or Ulk2 pre‐micro RNAs (miR) were electroporated into the tibialis anterior (TA) muscle of one leg, and a control miR plasmid into the contralateral leg of wild type mice. Muscles were harvested 7–8 days afterwards, either at basal conditions or after 24h of starvation. Second, ULK2fl/fl differentiated primary mouse myotubes were transduced with Ad‐Cre‐GFP or Ad‐GFP (control) viruses, and harvested up to 96h afterwards. Our results revealed that ULK2 is highly expressed in skeletal muscle, and that deficiency of ULK2, but not of its close homolog ULK1, leads to accumulation of ubiquitinated proteins. In addition, the autophagy receptors p62 and NBR1, which are degraded by autophagy and serve as receptors for ubiquitinated cargos, accumulate in muscles and primary myotubes deficient only in ULK2, independent of lysosomal fusion and function. Of note, the levels of the autophagy receptor optineurin were unaffected under the same conditions. These results indicate a novel and fundamental role for ULK2 in regulating p62‐ and NBR1‐dependent selective autophagy in skeletal muscle revealing ULK2 as a potential therapeutic target for skeletal muscle contractile and metabolic dysfunction.Support or Funding InformationSupported by AHA (16SDG30360001) and Dept. of Health & Human Physiology, University of Iowa.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Autophagy Enhances Protective Immunity of Macrophages Against Mycobacterium Tuberculosis
Authors: Lv Jingzhu , Wei Chunfang , Fang Fang , Zhang Yao , Kelly Gabriel , Wang Ting , Qian Zhongqing 
Published: June 2021

Autophagy has been considered as an immune mechanism in the last decade, and the involvement of autophagy in individual immune events is being investigated. More recently, autophagy has been linked with innate and adaptive immune responses against Mycobacterium tuberculosis (M. tuberculosis or TB), which can survive within macrophages by blocking the process of lysosome fusion with phagosomes. In addition, previous reports involving M. tuberculosis infection models showed the polarization of Type 1 T helper (Th1) cells in response to IL‐12 production and subsequent induction of IFN‐γ, which are two key players in immunity to TB. We sought to better understand the regulatory role of autophagy in the function of macrophages against M. tuberculosis, and the involvement of Th1 cytokine responses in these events. We found that lipopolysaccharide (LPS), a component of Gramnegative bacteria including M. tuberculosis, and also a pro‐inflammatory mediator, induced autophagy in M. tuberculosis infected THP‐1‐derived macrophages or PMA (phorbol 12‐myristate 13‐acetate) primed THP‐1 macrophages [THP‐1(A)]. This autophagy activation in THP‐1 or THP‐1(A) by LPS is very similar to Rapamycin, a known autophagy inducer. These two autophagy inducers significantly increased Th1 cytokine IL‐12 and IFN‐γ production, as well as M. tuberculosis killing effects in THP‐1 or THP‐1(A) cells. However, the autophagy inhibitor 3‐MA significantly reduced LPS activated IL‐12/IFN‐γ production or M. tuberculosis killing effects in THP‐1 or THP‐1(A) cells. These data demonstrate that autophagy induced by LPS stimulation mediates the protective immune remodeling in macrophages against M. tuberculosis. In parallel, we have shown levels of IFN‐γ presenting peripheral blood mononuclear cells (PBMC) collected from TB patients were significantly reduced compared to healthy volunteers. Similar to results in THP‐1 cells, LPS and Rapamycin restored the levels of IFN‐γ presenting PBMC from TB patients, which could be further raised by 3‐MA treatment. Combined together, our data strongly suggest a critical regulatory role of macrophage autophagy in innate immunity against M. tuberculosis, with involvement of the Th1 cytokine response.Support or Funding InformationThis study was funded by International Science and Technology Cooperation Project‐Key Research and Development Program of Anhui Province 1604b0602026 to ZQ (to support the analysis and interpretation of the data), and National Natural Science Foundation of China 81570011 to TW (to support the analysis and interpretation of the data, and preparation of the manuscript).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Restoration of SERCA ATPase as an Intervention to Muscle Impairment Associated with Oxidative Stress
Authors: Qaisar Rizwan , Bhaskaran Shylesh , Ranjit Rojina , Premkumar Pavithra , Huseman Kendra , Sataranatarajan Kavithalakshmi , Van Remmen Holly 
Published: June 2021

Disrupted intracellular calcium homeostasis is associated with various chronic muscle pathologies including sarcopenia. In support of this, the activity of SERCA, the Sarcoplasmic Reticulum (SR) Ca+2‐ATPase, a membrane enzyme that returns calcium to the SR lumen following muscle contraction, is reduced 14% in muscle of old wild type mice. Here we report a 27% decrease in SERCA activity in gastrocnemius muscle from mice lacking the superoxide scavenger CuZnSod (Sod1−/− mice), a mouse model of elevated oxidative stress and accelerated sarcopenia. The mechanism coupling reduced SERCA function with muscle impairment is not known. We hypothesize that restoring SERCA function will attenuate loss of muscle mass and function in the Sod1−/− mice. To test this, 2 month old Sod1−/− mice were treated for 7 weeks with CDN1163, a novel allosteric SERCA activator that acts directly to increase SERCA ATPase activity (50 mg/kg, IP, 3 times per week). Treatment with CDN1163 increased gastrocnemius muscle mass by 8% in CDN1163‐treated versus untreated Sod1−/− mice. In addition, the 22% reduction in specific force measured in untreated Sod1−/− versus age‐matched wild type mice was completely restored by 7 weeks of CDN1163 treatment. CDN1163 also reduced the oxidative damage measured as F2‐isoprostanes in muscle tissues by 50% compared to untreated Sod1−/− mice. Collectively our findings suggest that reduced function of the SERCA pump contributes to muscle atrophy and reduced force generation in the Sod1−/− mice and the pharmacological stabilization of SERCA with CDN1163 may be a powerful tool to counter age‐ and oxidative stress‐associated muscle impairment.Support or Funding InformationThis research was conducted while Dr. Rizwan Qaisar was a Diamond/AFAR award recipient and by a P01 grant (AG051442) to Dr. Holly Van Remmen.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The effect of the KATP channel opener Nicorandil in lipid‐peroxidation in skeletal muscle atrophy
Authors: PADILLA‐MALDONADO PAULINA , SANCHEZ‐DUARTE ELIZABETH , GÓMEZ‐BARROSO MARIANA , SANCHEZ‐PEREZ ALINA , CORTES‐ROJO CHRISTIAN , SAAVEDRA‐MOLINA ALFREDO , MONTOYA‐PEREZ ROCIO 
Published: June 2021

Muscle atrophy is defined as a decrease muscle tissue size due to cellular shrinkage caused by loss of organelles, cytoplasm and proteins. The main characteristic of this pathology is the increase in protein degradation and a decrease in protein re‐synthesis. It has been documented that the mitochondrial ATP sensitive potassium channel (mitoKATP), is involved in the resistance of the muscle fatigue and recovery from ischemia‐reperfusion injury and protection during atrophy process; Nicorandil has been described as a selective mitoKATP opener which confers protection to muscle during stressful conditions, the aim of this study was to determine the protective effect of Nicorandil over the damage caused by atrophy.C57BL/6 male mice 14–16 weeks‐old were used for the experimental basis; these animals were separated into 4 groups: 1) Control, 2) Nicorandil, 3) Atrophy and 4) Atrophy treated with Nicorandil. Atrophy was induced by hindlimb unloading by the tail method. Nicorandil was purveying in 40 mg/kg doses by intramuscular injection during 14 days. Once finished the treatment, the animals were sacrificed by cervical dislocation and the hindlimb were used for mitochondria isolation, once obtained the isoled mitochondria the lipid‐peroxidation was measured by the TBARS method. This reaction was measured in a Spectrophotometer Perkin Elmer Lambda 18 UV/VIS in ʎ of 523 nm. The results were for the group Nicorandil was higher against the Control group 50.5827%, the Atrophy group was 53.03% against the Control group, and the Nicrandil+Atrophy group had an increase in the activity against the Control group.Lipid‐peroxidation had been used as a marker of oxidative stress, as we know, this has been stablished that atrophy leads to an accumulation of toxic substances as reactive oxygen species, like we observe here the Nicorandil treatment has no effect in healthy mice but the opening of this channel downregulates lipid oxidation in the atrophied that can give us a thought of cytoprotection.Support or Funding InformationRMP‐CIC2017This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Activation of G Protein‐coupled Estrogen Receptor Promotes Endothelin‐dependent Natriuresis in Female Rats
Authors: Gohar Eman Y. , Obi Ijeoma E. , Daugherty Elizabeth M. , De Miguel Carmen , Kasztan Malgorzata , Pollock Jennifer S. , Pollock David M. 
Published: June 2021

The novel estrogen receptor, G protein‐coupled estrogen receptor (GPER), is a membrane‐bound receptor linked to acute signaling pathways. GPER activation elicits cardiovascular and nephroprotective effects against salt‐induced complications. However, its specific role in renal sodium handling is not yet defined. We hypothesized that activation of GPER in the renal medulla facilitates sodium excretion. Isosmotic saline was infused into the renal medullary interstitium (500 μl/h) of anesthetized male and female Sprague‐Dawley rats for an 80 min equilibration period and 20 min baseline urine collection period. Then, G1 (GPER agonist, 50 pmol/kg/min) or vehicle was infused into the renal medulla for an additional 20 min period. Compared with vehicle, G1 significantly increased urinary sodium excretion in females (from 0.6 ± 0.1 to 1.0 ± 0.1 μmol/min n=9, p<0.05). G1 did not change blood pressure or urinary potassium excretion in comparison with vehicle control (106 ± 3 vs. 105 ± 4 mmHg, 0.5 ± 0.1 vs. 0.4 ± 0.1 μmol/min, n=9). Medullary blood flow remained unchanged during these experiments. In males, G1 did not change urinary sodium excretion (from 0.6 ± 0.1 to 0.7 ± 0.1 μmol/min, n=9). No sex‐differences in medullary GPER mRNA expression was observed, suggesting that sex‐differences in G1 effects on sodium excretion could be related to GPER downstream signaling cascade. Given that endothelin‐1 (ET‐1) is a well‐established natriuretic factor that is regulated by sex and sex steroids, we hypothesized that GPER activation promotes natriuresis via an ET‐1‐dependent pathway. To test this idea, we studied the effect of medullary infusion of G1 after dual blockade of ETA and ETB receptors achieved by i.v. bolus injection of ABT‐627 (5 mg/kg) and A‐192621 (10 mg/kg), respectively. We found that the natriuretic response to medullary GPER activation in females was inhibited by dual blockade of ET‐1 receptors (from 0.5 ± 0.2 to 0.5 ± 0.2 μmol/min n=5). These data uncover a novel role for renal medullary GPER in promoting sodium excretion via an ET‐1‐dependent pathway in females, but not in males.Support or Funding InformationFunded by AHA 15POST25090329 to EYG and P01 HL136267 to DMP and JSPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SPAK disruption increases the glomerular filtration rate
Authors: Yilmaz Duygu Elif , Saritas Turgay , McCormick James , Thomson Martin , Bachmann Sebastian , Mutig Kerim 
Published: June 2021

The thick ascending limb (TAL) of the kidney reabsorbs approximately 25% of filtered NaCl via its luminal Na‐K‐Cl cotransporter (NKCC2). In specialized TAL cells constituting the macula densa (MD), NKCC2 serves to sense the luminal NaCl concentration for paracrine adjustments of the glomerular filtration rate (GFR) to the needs of the body. The catalytically active full‐length (FL) SPAK and OSR1 kinases activate NKCC2 by phosphorylation. In addition to the FL‐SPAK, TAL cells express truncated SPAK variants (SPAK2 and KS‐SPAK) arising from alternative splicing or proteolytic cleavage. SPAK2 and KS‐SPAK exert strong dominant‐negative effects on FL‐SPAK/OSR1‐dependent NKCC2 phosphorylation. This study addresses the role of SPAK variants in regulation of NKCC2 in MD cells with respect to the modulation of GFR.SPAK variants, OSR1, NKCC2 and phospho‐NKCC2 were analyzed in kidneys of wild type (WT) and SPAK‐deficient (SPAK−/−) mice and cultured TAL and MD cells using immunohistochemistry, immunoblotting and quantitative PCR. Key juxtaglomerular components involved in GFR regulation such as cyclooxygenase 2 (COX2), nitric oxide synthase 1 (NOS1) and renin were compared between the two mouse genotypes. Effects of SPAK deficiency on GFR were evaluated by transcutaneous assessment of FITC‐sinistrin clearance in freely moving mice.Immunohistochemical analysis revealed that NKCC2 phosphorylation levels were significantly higher in TAL cells of SPAK−/− kidneys compared to WT, supporting the presence of the dominant‐negative SPAK2 and KS‐SPAK isoforms in WT TAL cells. In contrast, NKCC2 phosphorylation was not significantly altered in SPAK‐deficient MD cells, suggesting minor effects of truncated SPAK variants on NKCC2 and a redundant role of FL‐SPAK in this cell type. In line with this, immunoblotting of lysates from cultured TAL and MD cells showed predominant abundance of SPAK2 and KS‐SPAK in TAL cells, whereas MD cells mainly expressed FL‐SPAK. Both TAL and MD cells showed substantial OSR1 levels, which may explain the FL‐SPAK redundancy. Juxtaglomerular COX2, NOS1 and renin levels as well as the FITC‐sinistrin clearance were substantially higher in SPAK−/− which might reflect increased NKCC2‐mediated salt reabsorption in preceding TAL and preserved NKCC2‐mediated sensing of luminal NaCl in MD cells.Our results describe differential expression of SPAK variants in TAL and MD cells and demonstrate that SPAK disruption stimulates GFR likely due increased NKCC2 activity in TAL prior to MD resulting in reduced NaCl concentration at the MD site.Support or Funding InformationFunding: DFG German Research Foundation Fellowship (332853055) and Else Kröner‐Fresenius Stiftung (2015_A197) to TS, R01DK098141 to JAM; DFG Grant MU2924/2‐2This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sorting nexin 27 regulates the lysosomal degradation of AQP2 protein in kidney collecting duct
Authors: Choi Hyo‐Jung , Park Eui‐Jung , Jang Hyo‐Ju , Cho Jeong‐In , Park Hye‐Jeong , Jung Hyun Jun , Kwon Tae‐Hwan 
Published: June 2021

We recently demonstrated that vacuolar protein sorting‐associated protein 35 (Vps35), a component of the retromer complex, interacts with the carboxyl terminus of aquaporin‐2, and depletion of Vps35 was associated with decreased AQP2 trafficking and increased lysosomal degradation of AQP2. Sorting nexin 27 (SNX27), a PDZ domain‐containing protein, is known to cooperate with the retromer complex in the recycling of early‐endosome to the plasma membrane. Since carboxyl terminus of aquaporin‐2 has the class I PDZ‐interacting motif (X‐Thr/Ser‐X‐Φ), we aimed to examine whether SNX27 could also play a role in the regulation of AQP2 trafficking and protein abundance in the kidney collecting duct cells. Immunohistochemistry revealed that SNX27 was diffusely labeled throughout the cytoplasm in the inner medullary collecting duct cells of rat kidney. Co‐immunoprecipitation assay of rat kidney inner medulla demonstrated that SNX27 interacted with AQP2. The role of SNX27 in the dDAVP‐responsive regulation of AQP2 trafficking and protein abundance was examined in mouse collecting duct mpkCCDc14 cells under siRNA‐mediated knockdown of SNX27. Cell surface biotinylation assay revealed that dDAVP‐induced translocation of AQP2 to the apical plasma membrane was decreased under SNX27 knockdown. Moreover, dDAVP‐induced AQP2 up‐regulation was significantly blunted in the cells with the siRNA‐mediated SNX27 knockdown. During the withdrawal period after dDAVP stimulation, the decrease of AQP2 protein abundance in the cells with SNX27 knockdown was attenuated by lysosomal inhibition with chloroquine co‐treatment. Taken together, SNX27 is an interacting protein with AQP2 and is likely to act as a component of the AQP2 sorting machinery after endocytosis, partly by regulating the lysosomal degradation of AQP2 protein abundance.Support or Funding InformationThis study was supported by the National Research Foundation of Korea Grants 2014R1A5A2009242 and 2016R1A2B4009365 funded by the Ministry of Science, ICT and Future Planning, KoreaThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.