bluuebunny/crossref_metadata_2025_split huggingface dataset:
Overexpression of miR‐503‐5p exacerbates hypoxia/reoxygenation injury in H9C2 cardiomyocytes
Authors: He Yanjing , Cai Yin , Liu Hao , Irwin Michael G , Xia Zhengyuan 
Published: June 2021

Acute myocardial infarction (MI) is a leading cause of death worldwide, and the most effective therapy for reducing acute myocardial ischemic injury is myocardial reperfusion, while reperfusion itself can lead to further cardiomyocyte death, called ischemia/reperfusion injury (IRI). A number of microRNAs have been shown to be involved in IRI, however, the role of miR‐503‐5p, which is implicated in cancers, diabetes mellitus or pulmonary arterial hypertension, remains unknown in myocardial IRI. This study was aimed to investigate the effects of miR‐503‐5p in myocardial IRI. Cardiac miR‐503‐5p expression was measured in mice subjected to myocardial IRI achieved by occlusion of left anterior descending artery for 30 minutes followed by reperfusion for 2 hours. H9C2 cardiomyocytes hypoxia/reoxygenation (H/R) was achieved by exposing the cells to glucose deprivation and hypoxia for 10 hours followed by reoxygenation 6 hours. We found that cardiac miR‐503 expression level was down‐regulated after induction of IRI compared with control, and it was also down‐regulated in H9C2 cells after exposure to H/R. In vitro, cell viability was decreased after H/R treatment, and lactate dehydrogenase (LDH) activity was increased. Administration of an agomir designed to overexpress miR503‐5p in cells before inducing H/R further reduced cell viability and elevated LDH activity. Moreover, proapoptotic Bax protein showed no significant change after overexpression of miR‐503‐5p in both normal and H/R states, while anti‐apoptotic Bcl protein decreased significantly. We analyzed putative miR‐503 targets using the TargetScan software, and found that the phosphoinositide‐3‐kinase regulatory subunit 1(PI3K p85) was one of the target genes. After overexpression of miR‐503‐5p, we found that PI3K p85 was decreased in both normal and H/R states. In addition, Akt1 and Akt2 as well as Phospho‐Akt were decreased in H/R state, and were further decreased after miR‐503‐5p overexpressed. Furthermore, we also found that prosurvival protein phosphorylation of signal transducer and activator of transcription (STAT3) was decreased after exposure to H/R, and declined further after overexpression of miR‐503‐5p. It is concluded that miR‐503‐5p regulates H9C2 cardiomyocyte injury during H/R, and exacerbates cell injury by inhibiting the activation of prosurvival protein Akt and STAT3.Support or Funding InformationSupported by Hong Kong RGC(17158616 M) and HMRF (05161826)grants.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Calibration and characterization of intracellular Asante Potassium Green probes, APG‐2 and APG‐4
Authors: Rana Priyanka S. , Gibbons Brandon A. , Alexy Vereninov A. , Yurinskaya Valentina E. , Clements Robert , Model Timothy M. , Model Michael A. 
Published: June 2021

The response of fluorescent ion probes to ions is affected by intracellular environment. To properly calibrate them, intracellular concentration of the measured ion must be made equal to its extracellular concentration. In the first, computational, part of this work, we show, using the example of potassium, that the two requirements for ion equilibration are complete dissipation of membrane potential and high membrane permeability for both potassium and sodium. In the second part, we tested the ability of various ionophores to achieve potassium equilibration in Jurkat and U937 cells and found a combination of valinomycin, nigericin, gramicidin and ouabain to be the most effective. In the third part, we applied this protocol to two Asante Potassium Green probes, APG‐4 and APG‐2. APG‐4 shows good sensitivity to potassium but its fluorescence is sensitive to protein density; therefore, calibration buffer had to be supplemented with 50 mM sucrose to keep cell volume constant. With these precautions taken, the average potassium concentrations in U937 and Jurkat cells were measured as 132 mM and 118 mM, respectively. The other tested probe, APG‐2, is non‐selective for cations; this is, however, a potentially useful property because the sum [K+] + [Na+] determines the amount of intracellular water.Support or Funding InformationThe research was partly supported by the Kent State University Research Council (MM) and by the Russian Fedration grant 0124‐2018‐0003 (VY and AV)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Heparin‐avidin interaction interferes with the XPerT permeability assay
Authors: Furman William , Garrett Joshua 
Published: June 2021

The endothelium is a monolayer of cells that line the vasculature and act as a semi‐permeable barrier controlling what passes from the lumen of blood vessels into the interstitium. Acute changes in endothelial permeability are commonly seen during inflammation. There are several in vitro assays which allow the researcher to look at changes in endothelial permeability under various inflammatory conditions, one of which is known as the XPerT assay. In this assay, endothelial permeability is assessed using fluorescence microscopy and is based on high affinity interactions between ligand (Alexa fluor‐488‐conjugated avidin) added in the culture medium, which permeates cell monolayer to reach substrate‐bound acceptor (biotinylated gelatin). Interestingly, heparin is known to strongly bind avidin and is also commonly found in different formulations of endothelial media in wildly differing concentrations. From this, we hypothesized that media with high concentration of heparin would interfere with the XPerT assay. To test this, we added increasing amounts of heparin to the XPerT assay and found that high doses impeded the ability of the avidin to interact with the biotinylated gelatin. Furthermore, we found that when high doses of heparin were present, the avidin was more likely to bind directly to the endothelial cells and not the biotinylated gelatin. Taken together, our data suggests that using a low heparin containing endothelial medium is optimal for using the XPerT assay to test endothelial permeability.Support or Funding InformationThis work was supported in part through the Hartwick College Faculty Research Grants Program.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Stromal Interaction Molecule and Gelsolin Mediate Pathological Cardiac Remodeling
Authors: Mancarella Salvatore 
Published: June 2021

Pathological cardiac remodeling is characterized by a series of poorly understood changes in myocardial microstructure involving changes in the cardiomyocytes cytoskeleton. Stromal interaction molecule 1 (STIM1) resides mostly in the sarco/endoplasmic reticulum where is senses intraluminal Ca2+ levels and activates the Orai channels on the plasma membrane to initiate Ca2+ influx. We have previously shown that STIM1 is involved in the dynamic remodeling of the actin cytoskeleton. However, the downstream effectors of STIM1 leading to cytoskeletal remodeling are not known. Proximity‐labeling technique (BioID) can capture weak and transient protein‐protein interactions. Hence, in the present study we investigated the STIM1 interactome used the BioID technique. A promiscuous biotin ligase BirA* was fused to the cytoplasmic C‐terminous of STIM1 and generated MEF stable cell line expressing STIM1‐BirA*. Biotin was added to the culture medium of MEF expressing STIM1‐BirA as well as in control cells not expressing STIM1‐BirA. Cells were harvested after twenty‐four hours and total lysates were incubated with streptavidin magnetic beads to pull down the biotinylated proteins which were subsequently identified by mass spectrometry. STIM1 interactors were validated in neonatal rat cardiac myocytes (NRCM) coupled with biochemical, immuno‐staining techniques and confocal microscopy. Screening of biotinylated proteins identified several high confidence known and previously unknown interactors of STIM1. One of the novel interactors was Gelsolin (GNS), a Ca2+‐dependent actin‐severing protein that is a key regulator of actin filament assembly and disassembly. STIM1 and GSN relationship was further investigated using NRCM. We show that STIM1 upregulation in NRCM was enough to induce upregulation of GNS protein levels. Immunostaining confirms colocalization between STIM1 and GSM. Furthermore, STIM1 overexpression in NVCM show altered actin dynamic and tubulin overexpression probably in response to cytoskeletal instability. We report the first use of BioID to characterize the STIM1 interactome. Our work unravels an unprecedented role of GSN as a part of the STIM1 interactome and its role in the modulation of the actin cytoskeleton.Support or Funding InformationWe gratefully acknowledge funding from the the National Heart, Lung and Blood Institute (award HL114869, to Dr. S.M.), and from UTHSC seed funding.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Decreased adiposity and enhanced glucose tolerance in Zyflamend treated mice
Authors: Alquraishi Mohammed , Donohoe Dallas , Voy Brynn H. , Whelan Jay , Bettaieb Ahmed 
Published: June 2021

Obesity is a growing epidemic that presents a major health problem worldwide. Obese individuals are at a higher risk of chronic diseases including cardiovascular diseases, nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes. Zyflamend is a poly‐herbal supplement derived from the extracts of ten different herbs that effectively activates AMPK in‐vitro in multiple cancer cell lines. When activated, AMPK is instrumental in inhibiting lipogenesis and enhancing catabolic pathways that generate ATP, such as fatty acid oxidation. The objective of this study is to investigate the effects of Zyflamend on adiposity and glucose homeostasis in vitro and in‐vivo. To that end, we fed mice chow, high fat diet, or high fat diet supplemented with Zyflamend over a period of 24 weeks and we examined alterations in body weight and glucose homeostasis. In addition, we treated human and mouse pre‐adipocytes with Zyflamend and examined changes in adipogenesis and key signaling pathways were determined in vitro. Mice fed High fat diet supplemented with Zyflamend displayed decreased weight gain and resistance to HFD‐induced glucose intolerance. Further, Zyflamend treatment decreased HFD‐induced hepatic dyslipidemia. These findings correlated with decreased adipogenesis of mouse and human adipocytes in‐vitro. Additionally, Zyflamend enhanced adipose insulin signaling as evidenced by increased tyrosyl phosphorylation of the insulin receptor and enhanced downstream signaling.ConclusionThese studies identify Zyflamend as a potential regulator of systemic glucose tolerance, energy balance and adiposity.Support or Funding InformationThe work was supported by NIH R00DK100736 (AB)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hepatic ZIP8‐mediated selenium transport contributes to normal liver function
Authors: Liu Liu , Liu Zijuan 
Published: June 2021

ZIP8 is a multi‐functional electroneutral membrane transporter with substrates including both essential and toxic divalent metals (e.g. zinc, manganese, iron and cadmium) and micronutrient selenite. Despite that ZIP8 is found to be associated with metabolic disorders by GWAS, the physiological and pathological roles of ZIP8 remain to be characterized and its connection to any metabolic pathways has not been identified. Our preliminary results demonstrate that moderate or acute decreases in ZIP8 activity resulted in hepatic pathology. Chronic moderate ZIP8 deficiency in transgenic mice led to spontaneous liver neoplastic nodules with increased oxidative stress, inflammation and fibrosis. And acute ZIP8 decrease in mice via adenovirus induced significant hepatomegaly, injury, inflammation and proliferation in liver. In addition, we found that elevation of ZIP8 induced significant glycogen accumulation while downregulation of ZIP8 decreases the glycogen level in primary hepatocytes and liver. Along with these liver pathology, the selenium level and selenoproteins (including GPX1 and GPX2) significantly decreased after ZIP8 deficiency. Thus ZIP8 plays an important role in maintaining normal hepatic function, likely through regulating selenium homeostasis.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Diverse Regenerative Strategies of the Intestinal Epithelium
Authors: Klein Ophir 
Published: June 2021

Epithelial surfaces form critical barriers to the outside world and are continuously renewed by adult stem cells. Whereas epithelial stem cell dynamics in homeostasis are increasingly well studied, how stem cells are redirected from a tissue‐maintenance program to initiate tissue repair after injury remains incompletely understood. Our initial work focused on the interrelationship between stem cell populations in the gut, and in this talk I will present more recent studies examining the response of the intestinal epithelium to damage. Using a parasitic helminth that disrupts tissue integrity during its life cycle as a model, we found that tissue damage leads to re‐initiation of a developmental program, thus representing a fundamental and novel mechanism by which the intestinal crypt can remodel itself to sustain function after injury. I will also discuss our experiments using live imaging of adult intestinal organoids, which show that nascent daughter dispersal occurs when neighboring cells insert between them during cytokinesis. Our data suggest that the mechanics of progenitor cell division provide a driving force for cell mixing in elongated mammalian epithelia.Funding provided by NIH and CIRM.Support or Funding InformationFunding provided by NIH and CIRM.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Conformational Resilience of Protein Disulfide Isomerase
Authors: Guyette Jessica , Evangelista Baggio , Tatulian Suren A. , Teter Ken 
Published: June 2021

Protein disulfide isomerase (PDI) is a 57 kDa protein with both oxidoreductase and chaperone activities that is mainly localized to the endoplasmic reticulum. It is a U‐shaped protein with an abb'xa’ structural organization where the a and a′ domains have conserved CGHC active sites, the b and b′ domains are involved with substrate binding, and x is an unstructured linker. PDI exhibits substantial structural flexibility and undergoes cycles of unfolding and refolding in its interaction with cholera toxin, suggesting PDI can regain a folded, functional conformation after exposure to stress conditions. To determine whether the unfolding‐refolding cycle of PDI is a substrate‐induced process or an intrinsic physical propensity of PDI, we used circular dichroism to examine the structural properties of PDI subjected to thermal stress. PDI exhibited remarkable conformational resilience that is linked to its redox status. In the reduced state, PDI exhibited a 54°C unfolding transition temperature (Tm) and regained 84% of its native structure after 70% thermal denaturation. Oxidized PDI had a lower Tm of 48°C and only regained 44% of its native conformation after 70% denaturation. Additional studies documented increased thermal stability of a PDI deletion construct lacking the a′ domain and decreased thermal stability of a construct lacking the a domain. Both constructs were able to regain a portion of their native conformation after exposure to thermal stress and, like full‐length PDI, were more stable in the reduced form than the oxidized form. A bb'x PDI construct lacking both a and a′ domains was highly stable, with a 60°C Tm in the reduced state and a 56°C Tm in the oxidized state. Both reduced and oxidized forms of the bb'x construct could return to the native state after heating to 90°C. These findings provide new insight into the physical foundation of the biological function of PDI.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hsp90 Recognizes a Conserved Motif in the A Chains of ADP‐Ribosylating Toxins that Move from the Endoplasmic Reticulum to the Cytosol
Authors: Kellner Alisha , Teter Kenneth 
Published: June 2021

Cholera toxin (CT) enters the cell via receptor‐mediated endocytosis and travels in a retrograde fashion to the endoplasmic reticulum (ER). The catalytic A1 subunit is then displaced from the rest of the holotoxin, unfolds, and is exported to the cytosol where it regains an active conformation for the ADP‐ribosylation of its G‐protein target. We have shown that the cytosolic chaperone Hsp90 is required for CTA1 translocation to the cytosol. We have also shown that Hsp90 is able to independently bind and refold CTA1. Using libraries of CTA1‐derived peptides, we have identified two distinct Hsp90 binding sites within the 192 amino acid protein, an N‐terminal RPPDEI (CTA111–16) motif and a C‐terminal LDIAPA (CTA1153–158) motif. Peptides corresponding to either site were able to block Hsp90 from binding to full‐length CTA1, and both binding sites were required for translocation of CTA1 to the cytosol. The LDIAPA motif is unique to CTA1, but an RPPDEI‐like motif is present in four other ER‐translocating ADP‐ribosylating toxins: pertussis toxin, Pseudomonas aeruginosa exotoxin A, Escherichia coli heat‐labile toxin, and Salmonella Typhimurium ADP‐ribosylating toxin. Using site‐directed mutagenesis to further investigate the RPPDEI motif, we found that a modification of either proline residue blocks CTA1 translocation to the cytosol. Our work has identified, for the first time, specific amino acid sequences that are recognized by Hsp90 and are essential for toxin translocation from the ER to the cytosol.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cryo‐EM analysis of full‐length receptor tyrosine kinase
Authors: Bai Xiaochen 
Published: June 2021

Receptor tyrosine kinases (RTKs) represent a family of cell surface receptors that have been shown not only to play a key role in regulating normal cellular processes, but also to be critical in the development and progression of many types of human cancer. To date, many crystal structures of individual domains of RTKs are available; however, the high resolution structural information for full‐length RTK proteins either in their apo‐state or in ligand‐bound states remains elusive. In addition, there are no structural detail about how the active full‐length receptor can bind the effector protein for the activation of downstream signaling pathway. Lacking a complete structure of full‐length receptor signaling complex hinder our understanding of how this important protein functions. We determine the structures of full‐length RTK proteins, in complex with their ligands and downstream proteins, by single particle cryo‐EM, as key to understand the critical role that this protein family plays in the physiology of the cell. Epidermal growth factor receptor (EGFR), RET, cMET and Insulin receptor (IR), the most studied members in the RTK family, are chosen as the targets for structure determination. These pathways comprise a complex and diverse signaling network, and play an essential role in normal cell proliferation, differentiation, development and motility. Aberrant activation can lead to both tumor growth and metastatic progression of cancer cells, which makes them promising candidate for cancer therapy. Determination of full‐length RTKs in apo‐ and ligand‐bound states shed light on its mechanisms of kinase activation, enhance understanding of the interaction between receptor and their ligand and inhibitors, and facilitate development of selective anti‐cancer drugs. Reconstitution of fully assembled signaling complex reveal the mechanism by which the active receptors initialize the downstream signaling transudation, and provide direct structural evidence for how the plasma membrane plays roles in this processing. Solving the structures of different members of the RTK family reveal similarities and differences in the structure‐function relationships within this receptor family, which can potentially lead to new strategies to selectively inhibit/modulate individual pathways for cancer therapy or concurrently target various pathways, an approach that has been suggested to improve therapeutic effectiveness.Support or Funding Informationthe Welch foundation, and CPRIT foundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cholesterol sulfate alters astrocyte metabolism and offers some neuroprotective effects
Authors: Prah Jude k , Yang Shaohua 
Published: June 2021

IntroductionCholesterol sulfate (CS) is one of the most important known sterol sulfates in human plasma and present as a normal constituent in a variety of human tissues. In both the brain and periphery, CS serves as a substrate for the synthesis of sulfonated adrenal steroids such as pregnenolone sulfate and Dehydroepiandrosterone (DHEA) sulfate and as a constituent of many biological membranes including red blood cells where it functions as a stabilizing agent. It also acts as endogenous regulator of cholesterol synthesis. It is known that CS serves as a substrate for synthesizing other sterol sulfates in the brain. However, the role of CS in neurological insult and brain metabolism is unknown. Our goal in this study is to investigate the neuroprotective action of CS as well as its effect on brain energy metabolism.Materials and MethodsPrimary astrocytes were prepared from the cortex of postnatal day 0–2 C57BL/6 pups and seeded in Dulbecco's modified eagle medium (DMEM) with 10% FBS under normal glucose (5.5 mM). HT‐22 cells were maintained in high glucose (25 mM) DMEM supplemented with charcoal stripped FBS. The neuroprotective effect of CS and its role on cell metabolism were determined in primary astrocyte and HT‐22 cells using Calcien AM cell viability assay, flow cytometry, seahorse extracellular flux analysis, and metabolism assay kits.ResultsCS protects HT22 cells against glutamate toxicity and impact astrocyte metabolism by increasing ATP, and glycogen contents.ConclusionOur study demonstrated that CS have neuroprotective effect and modulate brain energy metabolism. Further studies are needed to determine the mechanisms underlying the neuroprotective action of CS and its action on brain energy metabolism.Support or Funding InformationThis work was partly supported by National Institutes of Health grants 1R21NS087209‐01A1 (SY) and R01NS088596 (SY).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.