bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of Cl−in signaling and ion transport by IRBIT‐mediated recruitment of multiple kinase and phosphatase pathways
Authors: Vachel Laura , Shcheynikov Nikolay , Yamazaki Osamu , Fremder Moran , Ohana Ehud , Son Aran , Shin Dong Min , Yamazaki‐Nakazawa Ai , Yang Chin‐Rang , Knepper Mark A. , Muallem Shmuel 
Published: June 2021

IRBIT is a multifunctional protein that also controls the activity of IP3 receptors and multiple Cl− and HCO3− transporters, including NBCe1‐B, CFTR and Slc26a6, by unknown mechanisms. With the ubiquitous NBCe1‐B, IRBIT interacts with the autoinhibitory domain (AID), exposing two cryptic GXXXP motifs to confer regulation by Cl−in. Here, we report that IRBIT‐dependent combinatorial dephosphorylation of S232/S233/S235 controls NBCe1‐B close and open states by determining autoinhibition and regulation by Cl−in. The pS233/pS235 conformer is fully active, no longer activated by IRBIT or inhibited by Cl−in. The pS232/pS235 conformer is similar to wild‐type NBCe1‐B, while the pS232/pS233 conformer is partially active, but retains inhibition by Cl−in. IRBIT then recruits the kinases SPAK and CaMKII to control phosphorylation of S65 and S12, respectively, which determine inhibition by Cl−in at the 32GXXXP36 and 194GXXXP198 motifs. Recruitment by IRBIT of PP1 and calcineurin dephosphorylates S65 and S12, respectively, and relieves inhibition by Cl−in. These findings reveal how multiple kinase/phosphatase pathways use multiple phosphorylation sites to fine tune a transport function, which has important implications for epithelial fluid and HCO3− secretion.Support or Funding InformationAll the work was supported by NIH fundingThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acute Alcohol Exposure‐Induced let‐7a Inhibition Exacerbates Hepatic Apoptosis by Regulating Caspase‐3 in Mice
Authors: Beane Kaleigh , Pan Jeong Hoon , Kim Hyunjin , Tang Jingsi , Park Jeen‐Woo , Kong Byungwhi C , Shin Eui‐Cheol , Lee Jin Hyup , Kim Jae Kyeom 
Published: June 2021

Alcohol consumption is a critical risk factor for hepatic pathogenesis, including alcoholic liver diseases (ALD), but implications of alcohol induced dysregulation of microRNA (miRNA) in ALD pathogenesis are poorly understood. In this study, we aimed to explore effects of dysregulated miRNA profile on ALD conditions in mice. C57BL/6J male mice were treated with either saline (CON; oral gavage; n=8) or alcohol (EtOH; 3 g/kg·body weight; oral gavage; n=8) for 7 days. The ALD conditions were assessed by a serum AST/ALT ratio and hepatic fat accumulation. A total of 599 miRNA, and 158 key mRNAs of fatty liver and hepatotoxicity pathway were measured in liver tissues using NanoString miRNA expression array and quantitative real‐time PCR arrays, respectively. The array datasets were utilized to predict interactions with miRNAs. The predicted interactions of miRNA‐mRNA pairs were further validated using in vitro miRNA transfection experiments using AML12 mice hepatocytes. As results, miR‐21 and let‐7a were significantly decreased in EtOH group, and Ccng1, Rb1, and Casp3 mRNAs were predicted as their target genes. In addition, p53 expression was increased in the EtOH group, which is consistent with increased Casp3 activity, cPARP expression, and TUNEL positive cells by alcohol treatment. Similarly, in vitro suppression of let‐7a increased apoptosis by upregulating Cas‐3 protein expression in AML12 hepatocytes. Although there was no direct interaction between let‐7a and Rb1 protein expression, MDM2/p53 axis was also influenced by let‐7a inhibition. Therefore, alcohol‐induced inhibition of let‐7a may increase hepatic apoptosis by both directly targeting Casp3, and indirectly modulating MDM2/p53 axis.Support or Funding InformationThis work was supported by the University of Arkansas, VPRED Start‐up fund (JKK, and JHP). Support has been provided in part by the Arkansas Biosciences Institute, a partnership of scientists from Arkansas Children's Hospital, Arkansas State University, the University of Arkansas‐Division of Agriculture, the University of Arkansas, Fayetteville, and the University of Arkansas for Medical Sciences. The Arkansas Biosciences Institute is the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000 (JKK, JHP, KEB, and BCK). This work was also supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. NRF‐2015R1C1A1A01053746; JHL) as well as the China Scholarship Council (JT).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Long‐Term High‐Fat Diet Decreases Hepatic Iron Storage Associated with Suppressing TFR2 and ZIP14 Expression in Rats
Authors: Jiang Shuxia , Yan Kai , Sun Bo , Gao Shixing , Yang Xiaojing , Ni Yingdong , Ma Wenqiang , Zhao Ruqian 
Published: June 2021

High fat (HF) diet‐induced obesity is known to disturb hepatic iron metabolism in a time‐dependent manner. The mechanism of decreased hepatic iron store induced by long‐term high fat diet remains unclear. In this study, twenty‐four 6‐week‐old male Sprague‐Dawley rats were fed a high fat diet for 16 weeks and hepatic iron metabolism was examined. HF diet feeding considerably decreased hepatic iron contents, enhanced transferrin (TF) expression, reduced ferritin heavy chain (FTH), ferritin light chain (FTL), transferrin receptor 2 (TFR2) and ZRT/IRT‐like protein 14 (ZIP14) expression in the liver of rats. Down‐regulation of hepatic TFR2 coincided with DNA hypermethylation on the promoter and repressing expression of transcription factor hepatocyte nuclear factor 4α (HNF4α). MiR‐181 family expression was markedly increased and verified to regulate Zip14 expression using the dual‐luciferase reporter assay. These results indicate that the long‐term high fat diet decreases hepatic iron content, which is closely associated with inhibition of hepatic iron uptake via TFR2 and ZIP14‐dependent pathway.Support or Funding InformationThis work was supported by grants from the National Key Research and Development Program of China (2016YFD0500500), the National Natural Science Foundation of China (31302053), the Priority Academic Program Development of Jiangsu Higher Education Institutions, and the Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Combination of Traction Force Microscopy with Adhesive Protein Micropattern to Measure E‐cadherin‐based Intercellular Adhesion
Authors: Lu Chuan‐Fen , Tsou Chieh‐Chung , Chu Yeh‐Shiu , Lo Chun‐Min 
Published: June 2021

Cellular mechanical forces can be generated intrinsically and extrinsically; these physical cues regulate a wide variety of critical cellular functions such as cell proliferation, differentiation, and migration. Mechanical forces generated by cell‐cell interaction are able to transmit to the interior of the cell through the coordination of cortical actomyosin networks. Following that, physical signals can be converted to chemical ones through transducing process. E‐Cadherins (E‐Cdh) are prototypical adhesive molecules which can construct strong adhesive interfaces between cells. Currently E‐Cdh complexes comprised of β‐catenin and α‐catenin are suggested to associate with cortical F‐actin in a dynamic manner; however, the mechanistic steps of mechanotransduction in E‐Cdh‐mediated cell adhesion remain elusive. In this study, we attempt to establish a novel protocol for measuring intercellular forces generated by two adhering cells on 2D flexible substrate with a better temporal and spatial control. A combination of protein micropattern and traction force microscopy (TFM) is therefore developed. Pair micropattern is photolithographed on polyacrylamide gel (Young's modulus = 9 kPa) with two triangle forms, spaced 8 μm apart, to confine cell spreading area (1600 μm2). EpH4 cells of mouse breast epithelial origin are allowed to form the cell doublets on micropattern and then calcium switch is proceeded to initiate the E‐Cdh mediated cell adhesion. Pilot study has demonstrated that the intercellular strengths are measureable with calcium sensitivity and cadherin specificity. A series of CRISPR clones targeted primarily on E‐Cdh and p120‐catenin are prepared to decipher the novel mechanotransduction pathway of E‐Cdh. Thus, we describe an integrated mechanobiological method for the analysis of intercellular forces, which may provide deep insights on the biophysical principle governing mechanotransduction of E‐Cdh‐mediated intercellular adhesion.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanisms of DNA ligation
Authors: O'Brien Patrick 
Published: June 2021

Humans have three DNA ligases with distinct structural and biochemical features. Together, these enzymes are responsible for completing the majority of replication, repair, and recombination pathways. Deficiencies in the different enzymes cause different diseases and engineered cell lines lacking DNA ligases individually or in combination have challenged traditionally assigned roles of these enzymes. Quantitative mechanistic approaches with reconstituted enzymes and defined DNA breaks provide new insights into the substrate specificity and catalytic mechanisms of the different ligases that helps to define their normal biological roles and their compensatory roles when the other enzymes are compromised. These findings provide insight into the genomic instability of cancer cells and the immune deficiencies associated with defects in DNA ligases.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determining the Physiological Basis of No‐Go Decay Using sod1Δ Strains in Saccharomycescerevisiae
Authors: Lobb Amber , Cianflone Emily , Champine Madison , Bailey Lindsey , Seaberg Lucas , Segal Scott P 
Published: June 2021

Chromium VI (Cr(VI)) induces oxidative stress in cells, resulting in altered gene expression, increased apoptosis and cell death. Previous work showed that Cr(VI) exposure results in the formation of 8‐oxo(G) bases in mRNA. The presence of 8‐oxo(G) bases can lead to ribosome stalls during translation and activation of no‐go decay. During no‐go decay, the Dom34p/Hbsp1p complex acts to remove stalled ribosomes and subsequent endonucleolytic cleavage of the damaged mRNA at the stall site. Although oxidative agents and other non‐physiological substrates have been used to activate no‐go decay, very little work exists to elucidate the true physiological role of this pathway.Sod1p (superoxide dismutase 1) acts to scavenge free oxygen species by catalyzing the partitioning of superoxide (O2− ) into O2 and H2O2. Cells that lack functional Sod1p were observed to have increased oxidative stress resulting in increased P‐body assembly. Yet, global translation was only attenuated. To determine whether no‐go decay is being activated in strains lacking Sod1p, double mutant strains were created in which SOD1 was knocked out in conjunction with one of the effectors of no‐go decay. Consistent with no‐go decay being activated in response to oxidation, the hbs1Δsod1Δ strain resulted in a decrease in P‐body assembly, as compared to wild‐type, even in the presence of Cr (VI).Interestingly, Stm1p is thought to potentially to aid in the dissociation and recycling of the ribosomal subunits. In the stm1Δ strain, P‐body assembly was mildly decreased as compared to the wild‐type upon treatment with Cr(VI). This is consistent with Stm1p functioning to recycle ribosomes downstream of no‐go decay. Additionally, a stm1Δ can can suppress sod1Δ growth defects on Cr(VI) containing media.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Versatile Tools Toward Real‐time Single‐molecule Biology
Authors: Barbetsea Kassandra , Simpson Trey , Raja Ali , Candelli Andrea 
Published: June 2021

Biological processes performed by proteins interacting with and processing DNA and RNA are key to cell metabolism and life. Detailed insights into these processes provide essential information for understanding the molecular basis of life and the pathological conditions that develop when such processes go awry.The next scientific breakthrough consists in the actual, direct, real‐time observations and measurements of the individual mechanisms involved, in order to validate and complete the current biological models.Single‐molecule technologies offer an exciting opportunity to meet these challenges and to study protein function and activity in real‐time and at the single‐molecule level.Here, we present our efforts for further enabling discoveries in the field of biology and biophysics using the combination of optical tweezers with single‐molecule fluorescence microscopy (C Trap™).We show the latest applications of these technologies that can enhance our understanding not only in the field of DNA/RNA‐protein interactions but also in the fields of molecular motors, protein folding/unfolding, cell membranes and genome structure and organization.These experiments show that the technological advances in hybrid single‐molecule methods can be turned into an easy‐to‐use and stable instrument that has the ability to open up new venues in many research areasThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Studying the Reductase‐Chaperone Activity of the Epoxidase Component of Styrene Monooxygenase
Authors: Ma Chen Patrick , Gassner George 
Published: June 2021

Styrene Monooxygenase (SMO) is a two‐component flavoprotein composed of a NADH dependent reductase (SMOB) and FAD specific epoxidase (SMOA) that catalyzes the enantioselective epoxidation of vinyl side‐chains of styrenes. The chemical versatility of SMO makes it a desirable biocatalyst for the environmentally friendly synthesis of pharmaceuticals and for the removal of styrene as a toxic contaminant of terrestrial and aqueous environments. Previous thermodynamic and kinetic studies from our lab and others have demonstrated that we can stabilize the reduced, SMO(FADred), peroxide, SMO(FADOOH) or hydroxyflavin, SMO(FADOH), intermediate states by establishing steady‐state reaction conditions in the presence of NADH alone, NADH and the substrate analog, benzene, or NADH and the substrate, styrene. In addition to its oxygenase function, SMOA has been demonstrated to have a chaperone‐function that stabilizes SMOB and greatly increases the catalytic half‐life of the reductase. The nature of this interaction remains an area of current investigation.In the present work, the protein‐protein interaction of SMOA and SMOB was evaluated by a series of pulldown assays under various conditions of catalytic turnover. In this approach, the N‐terminally histidine‐tagged SMOA (NSMOA) and SMOB protein components were prepared in buffer systems designed to stabilize the aforementioned intermediate states and loaded onto a miniature nickel (Ni2+)‐affinity column. After loading, the column was first washed with buffer containing 10 mM imidazole to remove non‐specifically bound SMOB and then washed with buffer containing 250 mM imidazole to remove SMO bound to the column by the N‐terminal histidine tag of NSMOA. The composition of eluted protein was evaluated by UV spectroscopy and polyacrylamide gel electrophoresis. We find that holoSMOB with oxidized FAD bound (SMOBFADox) passes directly through the column and does not significantly interact with NSMOA immobilized on the Ni2+ column. In the presence of NADH, FADred is efficiently transferred from SMOB to NSMOA bound to the Ni2+‐resin. Under reaction conditions designed to stabilize FADred, FADOOH, and FADOH intermediate states of SMOA, a significant fraction of the resulting apoSMOB is observed to be bound to NSMOA. The fraction of apoSMOB not retained by NSMOA on the column is observed to lose activity and aggregate in the absence of the chaperone‐like activity of SMOA with a half‐life of minutes. These results support a reaction model in which the components of SMO interact weakly with each other when oxidized FAD is bound to SMOB. A stronger binding interaction occurs between apoSMOB and the reduced, peroyiflavin, and hydroyflavin intermediates of NSMOA. These interactions serve to stabilize SMOB in a catalytically active state.Support or Funding InformationNIH GM008574This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Germ Cell Apoptosis and Oxidative DNA Damage in Testicular Ischemia Reperfusion Injury: Survivin’ the Lipoxygenases
Authors: Al‐Maghrebi May 
Published: June 2021

ObjectiveTesticular ischemia reperfusion injury (tIRI) is widely accepted as the underlying mechanism for the pathophysiology of testicular torsion and detrorsion (TTD) in young children that could increase the risk of male infertility. The aim of this study is to examine the role of the pro‐oxidative lipoxygenases (LOXs)‐induced testicular oxidative stress in the etiology of tIRI‐induced germ cell apoptosis (GCA) and oxidative DNA damage using its potent selective inhibitor nordihydroguaiaretic acid (NDGA).Materials and methodsSprague‐Dawley male rats (n = 36, 8 weeks old, 250–300g) were randomly assigned to three groups: Sham, unilateral tIRI and tIRI + NDGA (25 mg/kg). Rats underwent 1 hour ischemia followed by 4 hours reperfusion. Harvested testes were evaluated for spermatogenic damage by H&E staining, oxidative DNA damage by biochemical assays, GCA, and tissue immunoexpression of the three LOXs (LOX‐5, ‐12 and ‐15). The induction of GCA and the involvement of the apoptosis pathways survivin/ASK‐1/JNK and was investigated by immunofluorescence staining.ResultsOur data showed that tIRI caused disorganization of the seminiferous tubules layers and significant spermatogenic damage. Increased LOX immunoexpression but not mRNA expression was calculated as a result of tIRI. Augmented levels of LOX‐induced protein, lipid, and DNA oxidative damage were significantly elevated during tIRI. In addition, tIRI activated the ASK‐1/JNK signaling pathway, which was associated with the down regulation of survivin, an inhibitor of apoptosis. Selective inhibition of LOXs by NDGA diminished the above tIRI‐induced changes.ConclusionThe study outcomes implicates the role of LOXs in the generation of reactive oxygen species, which partly contributed to the tIRI‐induced oxidative damage that also led to spermatogenic damage, oxidative DNA damage and induction of the apoptosis pathway survivin/ASK‐1/JNK. This new finding provides a good understanding to the tIRI/TTD mechanism in the field of translational medicine worldwide.Support or Funding InformationKuwait Foundation for the Advancement of Science (KFAS), Kuwait – Project Code: P116‐13MB‐01.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SKP2 Inactivation Suppresses Cell Proliferation and Regulates AR/FOXA1 Expression in PCa Cells
Authors: Celada Sherly I. 
Published: June 2021

Increased levels of SKP2 (S‐phase kinase‐associated protein 2), an E3 ubiquitin ligase, is frequently seen in advanced stages of prostate cancer (PCa). Correlations between SKP2 and the ubiquitin‐mediated degradation of key cell cycle regulators such as p21 and p27, as well as between SKP2 and the regulation of androgen receptor (AR) activity is frequently observed in PCa progression. Despite these associations, the molecular mechanisms responsible for the proto‐oncogenic effects of SKP2 in PCa remain elusive. The transcription factor FOXA1 (Forkhead box protein A1) is known to induce AR activity, and by doing so cause the activation of AR target genes. Deregulation of the AR/FOXA1 complex has been found to contribute to the progression of PCa and castration‐resistant prostate cancer (CRPC). We hypothesized that SKP2 impacts the function of the AR/FOXA1 complex contributing to the progression of CRPC. Using shRNA technology, we established stable SKP2 knockdown PCa cell lines to investigate the effects of SKP2 on CRPC growth. Our results demonstrate that SKP2 plays a critical role in the regulation of AR and FOXA1 expression in CRPC cell lines. SKP2 knockdown increased AR and FOXA1 levels in C4‐2B and 22RV1 cells, two CRPC cell lines. Furthermore, SKP2 knockdown significantly inhibited cellular proliferation and restored sensitivity to the AR antagonist (MDV3100) in C4‐2B and 22RV1 cells. Combined exposure to the AR antagonist (MDV3100) and a SKP2 inhibitor significantly reduced cellular proliferation, a result of cell cycle arrest and cellular senescence in C4‐2B and 22RV1. Endogenous and Myc‐tagged SKP2 as well as Flag‐tagged FOXA1 immunoprecipitation displayed a physical interaction between SKP2 and FOXA1. In vivo ubiquitination assay was performed using HEK293T cells transfected with HA‐Ub constructs demonstrating a SKP2 E3 ubiquitin ligase function for FOXA1. Our findings present a potential SKP2‐AR/FOXA1 signaling pathway that may be targeted therapeutically to inhibit CRPC malignancy.Support or Funding Information2U54CA163066MVTCP CA163069This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural organization of the FtsLB complex of the bacterial divisome
Authors: Senes Alessandro , Condon Samson G.F. , Mahbuba Deena‐Al , Armstrong Claire R. , Diaz‐Vazquez Gladys , Craven Samuel J. , Cui Qiang 
Published: June 2021

In Escherichia coli, FtsLB plays a central role in the initiation of cell division, possibly transducing a signal that will eventually lead to the activation of peptidoglycan remodeling at the forming septum. The molecular mechanisms by which FtsLB operates in the divisome, however, are not understood. We present a structural analysis of the FtsLB complex – performed with biophysical, computational and in vivo methods – that establishes the organization of the transmembrane region and proximal coiled coil of the complex. FRET analysis in vitro is consistent with formation of a tetramer composed of two FtsL and two FtsB subunits. We predicted subunit contacts through co‐evolutionary analysis and used them to compute a structural model of the complex. The transmembrane region of FtsLB is stabilized by hydrophobic packing and by a complex network of hydrogen bonds. The coiled coil domain likely terminates near the critical Constriction Control Domain, which might correspond to a structural transition. Presence of strongly polar amino acids within the core of the tetrameric coiled coil suggests that the coil may split into two independent FtsQ‐binding domains. The helix of FtsB is interrupted between the transmembrane and coiled coil regions by a flexible Gly‐rich linker. Conversely, the data suggest that FtsL forms an uninterrupted helix across the two regions, and that integrity of this helix is indispensable for the function of the complex. The FtsL helix is thus a candidate for acting as a potential mechanical connection to communicate conformational changes between periplasmic, membrane, and cytoplasmic regions.Support or Funding InformationThe work was supported by National Institutes of Health Grant (NIH) R01‐GM0997522 and National Science Foundation (NSF) grant CHE‐1415910 and DMS‐1661900. S.G.F.C. and G.D.V. acknowledge support by the NLM training grant 5T15LM0073.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.