bluuebunny/crossref_metadata_2025 huggingface dataset:
Baroreflex Influence on Left Ventricle Energetics and Changes after Myocardial Infarct
Authors: Bras‐Rosario Luis , Apura Joao , Rocha Isabel , Tiago Jorge , Pinto Fausto , Sequeira Adélia
Published: June 2021

In the classic description the Baroreflex is a negative feedback mechanism that controls arterial blood pressure, as sensed by the baroreceptors. It is a key component of the Autonomic regulation of the circulation in normal conditions and during Myocardial Infarction.

As the Baroreflex influences the heart rate, cardiac contractility and vascular resistance, we hypothesised that it must influence the mechanical interaction between the left ventricle and the arterial tree as well as cardiac energy consumption and efficiency during Myocardial Infarction.

The Baroreflex was evaluated in 11 rabbits by partial occlusion of the common carotid artery before and after the induction of cardiac ischemia by the occlusion of the left anterior descending artery. The left ventricular pressure and left ventricular volume were measured with an intraventricular pressure and impedance catheter.

We designed a custom software to characterize beat by beat left‐ventricular and arterial physiologic properties. From this data, left‐ventricle end‐systolic elastance (Ees), arterial elastance (Ea) and the coupling ratio (Ea/Ees) were computed. We also calculated left‐ventricular stroke work (SW), total energy expenditure (PVA), mechanical efficiency measured by the ratio of the SW to its theoretical maximum (Qload) and cardiac energetic efficiency (CEE=SW/PVA).

In Basal conditions upon the application of the Baroreflex, the Ees, Ea showed an approximately similar increase which resulted in a constant Ea/Ees. SW and PVA increased while Qload and CEE remained similar. After Myocardial Infarction there is an alteration in the Baroreflex response that increases Ea and Ees differently which resulted in an increase in Ea/Ees ratio. SW and PVA increased while Qload decreased and CEE remained similar (Table). The main difference between Basal and Myocardial Infarct responses during the Baroreflex are the increase in Ea/Ees and the decrease in Qload.

In conclusion the Baroreflex is a control mechanism of the cardiovascular system which exerts a feedback modulation on the arterial pressure through a balanced increase in cardiac contractility and arterial elastance, that preserves both its mechanical and energetic efficiency in Basal conditions. After Myocardial Infarct the Baroreflex changes its behaviour and increases cardiac contractility and arterial elastance maintaining the energetic efficiency crucial during ischemia at the expense of a decrease in mechanical efficiency.

Support or Funding Information

Modelação Matemática e Computacional da Fisiologia Humana (2013) Fundação para a Ciência e Tecnologia EXCL/MAT‐NAN/0114/2012

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bio‐inspired micropattern modulates stem cell fate and directs well‐ordered tubular tissue formation
Authors: Liu Xiaohua
Published: June 2021

Many tissues in the body possess highly‐organized microstructures that are a prerequisite for their physiological functions. However, regeneration of the tissues with well‐ordered microstructures has long been a challenge due to unprecedented technology difficulties. Here, we report a unique maskless micropatterning technology to create bio‐inspired nanofibrous 3D matrices with highly‐organized microstructure for tubular tissue regeneration. Our micropatterning method is a laser‐guided, non‐contact, high‐precision, flexible computer programming of machining process that creates highly‐ordered tubules (2–5 μm) in the bio‐inspired 3D matrix. The tubular architecture presents pivotal biophysical cues to control dental pulp stem cell alignment, migration, polarization, and differentiation. More importantly, when using this hierarchical matrix as a template, we successfully regenerated functional tubular dentin (the main component of a tooth) in vivo. Our bio‐inspired micropattern approach represents a powerful avenue not only for the exploration of a range of fundamental biological questions, but also for the regeneration of functional tissues with well‐organized structures.

Support or Funding Information

This research is supported by NIH/NIDCR R01DE024979.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glycemic Index Determination of Plant‐ and Whey‐Based Protein Nutritional Shakes
Authors: Glynn Erin L. , Alamdari Nima , Wilson Michael , Ezatagha Adish , Jenkins Alexandra L. , Sharafi Mastaneh
Published: June 2021

Background

The glycemic index (GI) is used to classify carbohydrate‐containing foods and products according to their effect on postprandial blood glucose responses. Low GI products release carbohydrates more slowly and elicit a lower glycemic response than higher GI products. Accordingly, low GI diets may help with weight and general health maintenance. The purpose of this study was to determine and compare the glycemic index of 4 flavors of plant‐based protein shakes and 4 flavors of whey+plant‐based protein shakes (Shakeology®, Beachbody, LLC).

Methods

GI was determined using the standard ISO method (ISO 26642:2010). Ten subjects (7 male, 3 female) aged 44 ± 13 with a body mass index of 26.4 ± 2.3 kg/m2 participated in this open‐label, randomized, crossover study. Subjects consumed the designated test shakes or dextrose control in a randomized order (all 25 g available carbohydrate). Blood samples were collected at time −5 and 0 (baseline) and at 15, 30, 45, 60, 90, and 120 minutes after product consumption. GI was calculated by expressing each subject's glucose incremental area under the curve (AUC) for the test shake as a percentage of the same subject's mean response to the dextrose control tests. GI values were expressed using the glucose scale where dextrose = 100.

Results

The GI values of the tested plant‐based shakes were: Vegan Chocolate and Café Latte flavors = 36 ± 5, Vegan Strawberry and Vanilla flavors = 37 ± 6. The GI values of the tested whey+plant‐based shakes were: Chocolate and Café Latte flavors = 33 ± 5, and Strawberry and Vanilla flavors = 29 ± 5. All of the 8 shakes tested were classified as low GI based on standard classification of low GI ≤ 55, medium GI 56 – 69, and high GI greater than 70. In addition, blood glucose levels dropped 8% below baseline following dextrose ingestion, but were within 1% of baseline for all tested shakes.

Conclusion

All varieties of Shakeology evaluated tested as low glycemic index. GI values were not different between the plant‐based protein and the whey+plant‐based protein varieties.

Support or Funding Information

This study was funded by Beachbody, LLC. Testing was independently conducted by GI Labs, Inc., Toronto, ON, Canada.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An Evolutionary Conserved DNA Replication Stress Response in Planarian
Authors: Shamoon Ushna , Tirgar Reyhaneh , Akpati Lois , Sawyer Nathaniel , Nam Edward
Published: June 2021

S. mediterranea is a planarian with extensive regenerative ability which requires substantial cell division. Adult stem cells (SCs) divide many times over an organism's lifespan to renew tissues. The reduced capacity of adult SCs to regenerate old and damaged tissue results in aging. However, DNA replication stress occurs every cell division and must be resolved. From yeast to man, an evolutionary conserved DNA replication stress response (RSR) is essential for life. The RSR is a signal transduction pathway regulated by Ataxia‐telangiectasia and Rad3‐Related kinase (ATR) and Checkpoint 1 kinase (CHK1) to respond to replication stress. However, the RSR has not been established in planarian nor do we know if it functions in regeneration. We hypothesize that the RSR may be essential for regeneration. Preliminary results show reduced regenerative ability in the presence of hydroxyurea (HU), a replication stress inducing agent. However, low concentrations (2mM) did not attenuate regeneration, indicating that the RSR may be present and able to overcome replication stress. To test this, we treated amputated tails with HU, ATR inhibitor (ATRi), and a combination of both. In 2mM HU, tails regenerated similarly to control. We observed only a one day delay in regeneration with ATRi alone. However, ATRi and 2mM HU in combination prevented planarian regeneration completely. This synergistic effect suggests that ATR is significant in the response to replication stress during regeneration. To obtain genetic evidence, we are in the process of knocking down ATR as well as other RSR genes like CHK1, WRN, etc. Further determining the importance of RSR genes in planarian will elucidate the conserved components and allow us to understand how the RSR contributes to the incredible regenerative abilities in planarian.

Support or Funding Information

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of A Novel S‐adenosylmethionine‐dependent Methylase by Electron Paramagnetic Resonance and Mössbauer Spectroscopies
Authors: Radle Matthew , LaMattina Joesph , Booker Squire
Published: June 2021

Radical S‐adenosyl‐L‐methionine dependent methylases are divided into four classes (A–D) with only A through C being extensively characterized in vitro. Class D methylases separate themselves by purportedly utilizing methylenetetrahydrofolate in their catalysis‐. The gene product from Methanocaldococcus jannaschii, MJ0619, is the pioneer member of this class and is hypothesized to install methyl groups into C7 and C9 of methanopterin, a key coenzyme for C1 metabolism in methanogeneic and methylotrophic bacteria. By extracting folates from E. coli transformed with a plasmid containing the MJ0619 gene, Allen, et.al. were able to detect and characterize methylated substrates and determine that 7‐methylpterin and 6‐ethyl‐7‐methylpterin were produced in vivo. Feeding studies in which E. coli were grown in media containing C2H3‐ methionine or C2H3‐acetate suggested that MJ0619, unlike members of classes A‐C, does not use SAM, but rather methylenetetrahydrofolate as the methyl donor. Excitingly, MJ0619 harbors a second CX3CX2C motif hypothesized to ligate an additional [4Fe‐4S] cluster and when the middle cysteine of this motif is changed to an alanine only 7‐methylpterin is detected. Although it is suggested that the N‐terminal Fe/S cluster is involved in C9 methylation there is little in vitro evidence to support this function and no spectroscopic characterization to identify it as a [4Fe‐4S] cluster. Herein, we report the heterologous expression and isolation of MJ0619, a member of a class D methylases, and employ Mössbauer and electron paramagnetic resonance spectroscopies to characterize its Fe/S cluster(s) of MJ0619.

Support or Funding Information

Howard Hughes Medical Institute

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Curli Proteins CsgE and CsgF Inhibit the Aggregation of Human Islet Amyloid Polypeptide
Authors: Khan Neelam , Arlantico Megan , Feliciano Elizabeth , Nguyen Hannah , Jayasinghe Sajith
Published: June 2021

Curli, a type of proteinaceous cell surface filament, found on enteric bacteria such as E. Coli and Salmonella, are thought to play an important role in host cell adhesion and invasion. The assembly of CsgA, the major structural protein of Curli, into fibers is aided by three non‐structural proteins, CsgE, CsgF, and CsgG. CsgE and CsgF are thought to be periplasmic chaperone proteins that play a vital role in the assembly of Curli fibers. It has been shown that CsgE is able to prevent the aggregation of CsgA into Curli fibers. We determined the ability of CsgE and CsgF to inhibit the aggregation of human Islet Amyloid Polypeptide (hIAPP), an amyloidogenic protein unrelated to Curli formation. We monitored the aggregation of hIAPP, using the fluorescent dye ThT, in the absence and presence of CsgE and CsgF. An increase in ThT fluorescence emission intensity is routinely used to detect the aggregation of amyloidogenic polypeptides. In the absence of CsgE or CsgF we observe a greater than 5‐fold increase in fluorescence intensity when freshly dissolved hIAPP is monitored in the presence of ThT. Such an increase in intensity is typically taken to indicate the aggregation of hIAPP. In the presence of CsgE and CsgF no significant increase in ThT intensity is observed suggesting that both CsgE and CsgF prevented the aggregation of hIAPP.

Support or Funding Information

1. This work is supported by NIH grant 1R15GM123430‐01

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Induction of Neoplastic Transformation, AP‐1 Signaling, PDCD4 signaling, and DNA Damage by Copper Oxide Nanoparticles Involves ROS‐Mediated MAPK Pathways in JB6 Cells.
Authors: Barber Tabatha L. , Aldinger Joni , Tenney Allyson , Leonard Stephen , Morris Anna , Ding Min
Published: June 2021

Occupational exposures to copper dusts or fumes have beenreported to be harmful to human health, with possible risk of cancer amongcopper smelting workers. Copper (II)oxide (CuO) nanoparticles have not, to our knowledge, been extensively examinedfor potential carcinogenic or genotoxic effects. To investigate the mechanismsof CuO‐induced pathogenesis, the effect of CuO on AP‐1‐MAPKs and ROS generation were investigated. The results indicated that CuO caused a 2‐fold increase in AP‐1 activity in JB6 cells. The induction of AP‐1 activity in cultured cell lines was time and dose‐dependent. The signal transduction pathways for AP‐1 activation were also investigated. Western Blotanalysis demonstrate that CuO nanoparticles stimulate phosphorylation of p38MAPK and ERKs. CuO also generated ROS when incubated with the cells as measured by electron spin resonance (ESR). Nano‐sized CuO generated more ROS than the fine sized particles when incubated with the cells. COMET assay suggests that exposure of the cells to CuO resulted in DNA damage. Cells pre‐treated with ROS inhibitors sodium formate and Polyvinylpyridine‐N‐Oxide (PVPNO) before exposure to CuO nanoparticles demonstrate a significant reduction in p‐38 and ERK activity, suggesting the H2O2 may have a role in the ROS mechanism. Soft agar transformation assays demonstrated that there is a significant increase in colony formation in JB6/AP‐1 cells treated with CuO nanoparticles as compared to the control, indicative of neoplastictransformation. Unraveling the complex mechanisms associated with these events may provide insights into the initiation and progression of CuO‐induced pathogenesis.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Aggregated CsgA Does Not Form In‐Register Parallel Structures
Authors: Basham Michael , Guevarra Culbert , Meza‐Barajas Osmar , Godfrey Gabriel , Jayasinghe Sajith
Published: June 2021

Curli are hair‐like fibers used by the bacteria for adhesion and invasion, implicating them in the pathogenesis of bacterial disease. The main protein component of Curli is CsgA, and the main focus of our work is to characterize the structure of oligomerized CsgA. We expressed six C‐terminally histidine tagged single cysteine mutants of CsgA and used circular dichroism (CD) spectroscopy and pyrene fluorescence spectroscopic techniques to determine structural changes that occur during protein aggregation. CD spectra obtained immediately after addition of CsgA to buffer exhibit minima at approximately 200 nm, characteristic of a random coil conformation, indicating that CsgA is predominantly unstructured in solution. In contrast, CD spectra obtained after incubating CsgA in buffer exhibit minima at approximately 218 nm. These spectra are characteristic of β‐sheet conformation, indicating that CsgA undergoes a random coil to β‐sheet conformational change upon incubation. We also observed an increase in Thioflavin T fluorescence intensity upon incubation of CsgA in buffer indicating the formation of protein aggregates. Fluorescence spectra obtained of CsgA labeled at positions 24, 41, and 55 with pyrene did not contain a peak at 475 nm due to pyrene excimer formation, indicating that aggregated proteins are not arranged in an in‐register parallel fashion.

Support or Funding Information

1. This work is supported by NIH grant 1R15GM123430‐01

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Arsenic‐Induced Perturbations in Cholinergic System and Energy Metabolism of Rat Brain: Ameliorative effect of Vitamin‐E
Authors: Chinthirla BD , Konduru KK , Kozlova EV , Curras‐Collazo MC
Published: June 2021

Chronic exposure to inorganic arsenic compounds may lead to peripheral and central neurotoxicity. The aim of this study was to examine the impact of Arsenic (As) exposure on cholinergic and bioenergetic systems in three brain regions: cerebral cortex, cerebellum and hippocampus. Adult male Albino rats at 3 months of age were exposed to low (1.5 mg/kg body weight) and high dose As (3 mg/kg body weight) through intraperitoneal injection daily for a period of 3 weeks. A separate batch of rats were given α‐tocopherol (5mg/kg body weight) for a period of one week immediately after the low and high dose As exposure. The acetylcholinesterase (AChE) activity and acetylcholine (ACh) content in synaptosomal fractions and Mg2+‐ATPase and Na+/K+‐ATPase activities in mitochondrial fractions. We found that As treatment had decreased AChE, Mg2+‐ATPase and Na+/K+‐ATPase activities and increased in ACh content when compared to control (sham‐injected). The high dose As treated rats showed significantly decreased AChE, Mg2+‐ATPase and Na+/K+‐ATPase activities and increased ACh content compared to low dose As treated rats. The maximal alteration of these enzymes was observed in hippocampus among the three brain regions. Whereas, the rats supplemented with α‐tocopherol (along with As) showed a reversal effect with slight increase in enzyme activities and decrease in ACh content in a dose dependent manner. In addition to this, As exposure decreased mean body weights in a dose dependent manner. This study demonstrates that exposure to As provoked neurochemical and metabolic disruption by inducing alterations in enzymes of cholinergic system and energy metabolism. Moreover, Vitamin‐E treatment may have partial ameliorative effects on these disturbances caused by As toxicity.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determining Invertase Enzyme Activity Using Isothermal Titration Calorimetry
Authors: Chan Dindi , Anderson Jeremy , Kenealey Jason
Published: June 2021

Invertase catalyzes the hydrolysis of sucrose producing fructose and glucose. Invertase is widely used in the food industry because it possesses a lower crystallinity than sucrose at higher concentrations and is sweeter than sucrose. Invertase enzyme activity is typically measured using an endpoint coupled‐secondary reaction. The glucose produced by the invertase‐catalyzed reaction reacts with 4‐hydroxybenzoic acid hydrazide to produce yellow anions that can then be measured spectrophotometrically. Contrary to the standard method for measuring invertase activity, single injection isothermal titration calorimetry (ITC) directly measures heat generated by invertase continuously. ITC provides rapid and efficient measurement for enzyme activity. However, recent studies have shown that the single injection ITC measurements cannot separate km and kcat because the enzyme does not reach saturation. We recently demonstrated that single injection ITC can be used to measure the stability of invertase activity. Here we further characterize the relationship between the peak invertase activity and the peak heat‐rate. Additionally, previous ITC measurements of invertase activity have demonstrated that there is a delay in peak invertase activity. We elucidate the dependence of this delay in activity on the sucrose concentration. Single injection ITC is uniquely positioned to provide insight into the mechanistic action of invertase that cannot be determined using coupled reactions.

Support or Funding Information

Build Dairy

BYU MEG Grant

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Invasion and Inhibition in Cell Embryonic Migration
Authors: Kulesa Paul , McLennan Rebecca , McKinney M Cathy , Teddy Jessica M. , Morrison Jason A , Kasemeier‐Kulesa Jennifer C , Giniunaite Rasa , Robinson Martin , Baker Ruth , Maini Philip
Published: June 2021

The embryonic neural crest offers a unique model system in which to study cell migration mechanisms in vivo during development and disease. The neural crest is a highly invasive, multipotent cell population that displays a regulated spatio‐temporal migratory pattern in the vertebrate embryo. Importantly, several aggressive tumor types, including melanoma and neuroblastoma, originate from neural crest‐derived cells. This has led to the hypothesis that neural crest‐related malignancies may be intrinsically predisposed to increased metastatic potential due to the inherent invasive abilities of the neural crest. I will discuss our recent findings of novel neural crest invasion and inhibitory signals that sculpt the embryonic cell migration pattern and provide insight into limiting metastatic behaviors.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ube2v1, a ubiquitin‐conjugating enzyme variant, is an essential component for the ubiquitination of MEKK1 in FGF2‐mediated signaling in endothelial cells
Authors: Yoo Yung Joon , Elangovan Muthumumar , Jin Suk Won
Published: June 2021

Ubiquitin can be covalently conjugated to target proteins to provide a critical signal in diverse cellular processes. Given that ubiquitination is catalyzed by an E1‐E2‐E3 cascade of enzymes but the actual formation of peptide bond mostly occurs at the active site of E2, we decided to screen the mRNA level of E2s in the endothelial cells, intending to find the role of ubiquitination in angiogenesis. Based on our initial microarray data identified 33 E2s in human and its 25 paralogs in zebrafish, we analyzed the expression level of 25 E2s in endothelial cells of zebrafish embryos and found that Ube2v1, a ubiquitin‐conjugating enzyme variant, is the most highly expressed E2. The knockdown of Ube2v1 using siRNA significantly inhibited the proliferation and migration of human umbilical vein endothelial cells (HUVECs). It was also found that Ube2v1 was an essential component for FGF2‐mediated activation signaling by the ubiquitination of MEKK1 with K63‐linked ubiquitin chains. Interestingly, Ube2v1 was not involved in the VEGF‐A induced signaling in HUVECs. Although Ube2N is a canonical partner of Ube2v1 for K63‐linked ubiquitination, siRNA‐mediated knockdown of Ube2N had no effect, implying an alternative E2 partner for Ube2v1 for the ubiquitination of MEKK1.

Support or Funding Information

This work was supported by the GIST Resach Institute (GRI).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
More Efficient Breakdown of PET Plastic by Modifying the PETase Enzyme
Authors: Duplan Amanda , Hall Bonnie L.
Published: June 2021

Overproduction and improper disposal methods have caused the world's oceans and ecosystems to be polluted with plastic waste. Polyethylene terephthalate (PET) is a thermoplastic polyester resin widely used in plastic products. PET plastic contains a high ratio of aromatic components that are poorly reactive and difficult to degrade, so PET remains in the environment for an extensive time. In 2016, the novel bacterium Ideonella sakaiensis was discovered to be capable of using PET as a carbon source for growth. The enzyme PETase was isolated from the bacterium and found to break down PET plastic at a slow rate. Our goal is to modify the PETase enzyme to hydrolyze PET plastic with a higher efficiency. The over‐expression and purification of the PETase protein has been optimized. High performance liquid chromatography and nuclear magnetic resonance assays are being used to measure the rate and efficiency of PET breakdown. PETase with various amino acid substitutions has been generated, with the goal of improving enzyme activity under commercially relevant conditions. An optimized PETase enzyme capable of degrading these plastics efficiently on a large scale could be the first step to repairing our polluted ecosystems.

Support or Funding Information

This work was supported in part by the FUTURE program at the University of Iowa Carver College of Medicine.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Relationship between Furin Cleavage and Cytotoxicity in a Recombinant Immunotoxin Based on Pseudomonas Exotoxin A
Authors: Reifer Danielle , Grossman Brian , Weldon John E.
Published: June 2021

Recombinant immunotoxins (RITs) are anti‐cancer therapeutic proteins that combine an antibody and a protein toxin. RITs based on the bacterial toxin Pseudomonas exotoxin A (PE) are currently in clinical trials, with one (moxetumomab pasudotox) recently approved by the FDA for the treatment of Hairy Cell Leukemia. PE‐based RITs are internalized by receptor‐mediated endocytosis and undergo retrograde trafficking to the endoplasmic reticulum, from which they translocate to the cytosol and catalytically inhibit protein synthesis. Early on during their intracellular trafficking route, PE‐based RITs are hydrolyzed and activated by furin, a serine protease ubiquitously expressed in mammalian cells. The furin cleavage site in PE, however, has a very slow cleavage rate compared to other targets of furin. Because the furin cleavage site of PE is so poor, it has been proposed that altering the cleavage site to enhance the rate would increase the cytotoxicity of PE‐based RITs. Previous research has shown that furin site mutations can modestly enhance RIT activity, but no correlation exists between cleavage efficiency and cytotoxicity across the full site. Interestingly, the correlation becomes significant if only mutations in the N‐terminal region of the cleavage site are considered (Spearman's r = 0.986; p = 0.0056). To investigate this relationship further, three different forms of an anti‐transferrin receptor PE‐based recombinant immunotoxin have been expressed and isolated: one with mutations on the N‐terminal region of the cleavage site, one with mutations on the C‐terminal region, and one with mutations over the entire cleavage site. Each toxin will be refolded, purified by column chromatography, and evaluated in cytotoxicity assays against HEK293 cells to determine how they impact cytotoxicity. It is expected that the RIT with optimizing mutations across the entire furin cleavage site will exhibit the greatest increase in cytotoxicity, followed closely by the N‐terminal mutations alone. These data will provide a better understanding of the relationship between cleavage efficiency and cytotoxicity, permitting better design of PE‐based RITs to produce more effective cancer treatments. It will also help to elucidate an incompletely understood PE intoxication pathway.

Support or Funding Information

This project was supported by funds from the Linda Sweeting Summer Research Fellowship and the Fisher College of Science and Mathematics.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of RGS Homology and Pleckstrin Homology Domains in GPCR Kinase 2 Activation
Authors: Elkins Kaely Elizabeth , Cato M. Claire , Tesmer John J. G. , Sterne‐Marr Rachel
Published: June 2021

G protein‐coupled receptors (GPCRs) are transmembrane proteins that act through multi‐step pathways to regulate metabolic and synaptic signaling, as well as many other processes. GPCR kinases (GRKs) regulate GPCR signaling via phosphorylation. The seven vertebrate GRKs map to three subfamilies: GRK1 (GRK1/7), GRK4 (GRK4/5/6), and GRK2 (GRK2/3). GRK2 is a modular protein composed of an AGC kinase domain (KD), a pleckstrin homology domain (PHD) that binds Gβγ subunits and phospholipids, and a regulator of G protein signaling homology domain (RHD). The RHD is the central domain and makes contacts with both PHD and KD. Although GRK2 is activated by Gβγ and membrane phospholipids, it is its interaction with an activated GPCR that most dramatically promotes kinase domain activation. We seek to understand how GPCRs activate GRK2. Previously we used site‐directed mutagenesis to test the hypothesis that the release of inter‐domain contacts might artificially activate GRK2. Our hypothesis was not supported: mutations at the RH/KD small lobe interface disrupted the structural integrity of GRK2, while a mutation at the RH/KD large lobe interface (E96A) did not impact rhodopsin phosphorylation (Lodowski et al., 2005). This contrasts recent in vitro kinetic studies and molecular dynamic simulations with GRK4 family members (Komolov et al., 2017; Yao et al., 2017) that suggest that disruption of the RHD/KD interface (“ionic lock”) leads to RHD separation from the kinase domain and allows the KD to acquire an active conformation. The PH domain, only found in the GRK2 subfamily of GRKs, is essential for localizing GRK2 to the site of an activated receptor. Comparison of crystal structures of monomeric GRK2 versus the GRK2–Gβγ complex suggests that Gβγ binding causes slight rotation of the RHD away from the KD (Lodowski et al., 2005). Single time‐point kinase assays showed that mutations at the RHD/PHD interface, particularly P638D, impaired GRK2 function. It is possible that the PHD in GRK2 subfamily plays a large role in receptor mediated KD activation. In this work, we use an intact cell phosphorylation assay as well as more detailed kinetic studies in combination with expanded mutagenesis to assess the role of the RHD and the RHD/PHD interface in GRK2 activation.

Support or Funding Information

Siena College Center for Undergraduate Research and Creative Activity

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phosphorylation of yeast Pah1 phosphatidate phosphatase by casein kinase I
Authors: Hassaninasab Azam , Han Gil‐Soo , Carman George M
Published: June 2021

Pah1 phosphatidate phosphatase in Saccharomyces cerevisiae catalyzes the penultimate step (i.e. the production of diacylglycerol by dephosphorylation of phosphatidate) in the synthesis of triacylglycerol. The enzyme is subject to phosphorylation (e.g. cyclin‐dependent protein kinases Pho85‐Pho80 and Cdc28‐cyclin B, protein kinases A and C, and casein kinase II) and dephosphorylation (e.g. by the Nem1‐Spo7 phosphatase complex) for regulation of its cellular location, catalytic activity, and stability/degradation. In this work, we show that Pah1 is also phosphorylated by casein kinase I. By mass spectrometry, phosphopeptide mapping, and phosphoamino acid analysis, we showed that the phosphorylation occurs on multiple serine and threonine residues. Using Pah1 as substrate, casein kinase I activity was dependent on the time of the reaction and the amount of protein kinase, as well as the concentrations of Pah1 and ATP. The prephosphorylation of Pah1 by casein kinase I reduced its subsequent phosphorylation by Pho85‐Pho80, and the prephosphorylation by Pho85‐Pho80 reduced its subsequent phosphorylation by casein kinase I. This was attributed to both protein kinases having phosphorylation sites on Pah1 in common. The prephosphorylation by casein kinase I stimulated subsequent phosphorylation by casein kinase II, but the prephosphorylation with casein kinase II did not affect subsequent phosphorylation by casein kinase I. This indicated that the sites phosphorylated by casein kinases I and II were not common. Prephosphorylation of Pah1 by casein kinase I had no effect on the phosphorylation by protein kinase A, and vice versa. Hierarchal phosphorylation experiments with protein kinase C, along with experiments to identify sites on Pah1 phosphorylated by casein kinase II are currently in progress.

Support or Funding Information

GM050679

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
STX17 dynamically regulated by Fis1 induces mitophagy via hierarchical macroautophagic mechanism
Authors: Xian Hongxu , Yang Qiaoyun , Liou Yih‐Cherng
Published: June 2021

Mitochondrial quality control is essential for cellular homeostasis and accumulating evidence show that mitochondria can be selectively targeted for autophagic degradation (mitophagy). Mitophagy allows for the removal of dysfunctional mitochondria, which is highly implicated in energetically demanding cells including muscle and nerve cells. However, despite the well characterization of PINK1/Parkin route of mitophagy, mechanisms concerning PINK1/Parkin‐independent mitophagy are still poorly understood. With imaging approaches including structure‐illumination microscopy (SR‐SIM), here we demonstrate that the autophagy protein Syntaxin 17 (STX17), initiates mitophagy upon the depletion of outer mitochondrial membrane protein Fis1. Using mass spectrometry analysis, we identify STX17 interacts with Fis1, which preferentially gatekeeps the dynamic shuffling of STX17 between ER and mitochondria. Loss of Fis1 results in the accumulation of STX17 on mitochondria and mitochondria associated membranes (MAM), exposing its N‐terminus to assemble and self‐oligomerize for mitophagy. Mitochondrial STX17 interacts with ATG14 and further recruits core autophagy proteins hierarchically to form mitophagosomes, followed by Rab7‐dependent mitophagosome‐lysosome fusion. In addition, our results reveal that Fis1 loss impairs mitochondrial metabolic function, and potentially sensitizes mitochondria to STX17‐mediated mitochondrial engulfment within autophagosomes, which is directly initiated through canonical autophagy machinery, closely linking non‐selective macroautophagy and mitochondria. Our findings uncover a novel PINK1/Parkin‐independent mitophagy mechanism, in which outer mitochondrial membrane protein Fis1 gates the elimination of mitochondria.

Support or Funding Information

This work is financially supported by grants Tier 2 MOE and NUS, Singapore to Y.‐C.L.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of figs fruits extracts on rotenone‐induced Parkinson's disease‐like cytotoxicity in SH‐SY5Y cells
Authors: Essa Musthafa Mohamed , Al‐Adawi Samir , Manivasagam Thamilarasan , Justin‐Thenmozhi Arokiasamy
Published: June 2021

The current study was aimed to find out the effect of figs fruits extracts (FFE) on in vitro rotenone model of Parkinson's disease (PD) by evaluating redox status, mitochondrial membrane potential, cell viability, macromolecule oxidation, and apoptosis. Incubation of SH‐SY5Y cells with rotenone lead neuronal death, mitochondrial dysfunction and DNA fragmentation thorugh redox imbalance, condensation and apoptosis. Rotenone exposure also enhanced the expression of cytochrome C, caspase ‐3, ‐8, ‐9 and Bax (markers of apoptosis) and altered the anti‐apoptotic (Bcl‐2) markers. Reduction of the intracellular reactive oxygen species (ROS) levels ensued due to pretreatment of FFE along with improved antioxidant status, alleviation of rotenone induced mitochondrial dysfunction and apoptosis. Our current findings suggested that figs fruits extracts exhibit the marked neuro‐protective effects, which could be due to its antioxidant, mitochondrial protection and anti‐apoptotic properties. We thus conclude that FFE could be developed as a novel therapeutic molecule for the benefit of reducing the consequences of PD. However, further extensive preclinical and clinical studies are warranted.

Support or Funding Information

The funding support given by Sultan Qaboos University in the form of Internal grant (IG/AGR/FOOD/17/02) to Dr. Mohamed Essa is highly acknowledged.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Basis of Anthracycline Drugs Transport by P‐Glycoprotein
Authors: Nguyen Phuong H. , Sigdel Krishna P. , Kings Gavin M. , Roberts Arthur G.
Published: June 2021

P‐glycoprotein (Pgp) is an ATP‐dependent efflux transporter and plays a major role in anti‐cancer drug resistance by pumping a chemically diverse range of cytotoxic drugs from cancerous tumors. Despite numerous studies with the transporter, the molecular features that drive anti‐cancer drug efflux are not well understood. Even subtle differences in the anti‐cancer drug molecular structure can lead to dramatic differences in their transport rates. To unmask these structural differences, the focus of this study was on the closely‐related anthracycline drugs daunorubicin and doxorubicin. While only differing in the positions of their hydroxyl functional groups, daunorubicin has a 5‐fold higher transport rate than doxorubicin. They were both anticipated to activate Pgp‐mediated ATP hydrolysis, but instead they reduced Pgp‐mediated ATP hydrolysis below basal levels and to the same extent. This suggested that daunorubicin and doxorubicin mediated ATP hydrolysis of Pgp similarly. The KD values measured from from intrinsic tryptophan fluorescence of the transporter for both daunorubicin and doxorubicin are insignificantly different, implying that two drugs have similar affinity for the transporter. Interestingly, acrylamide quenching of Pgp tryptophan fluorescence revealed that daunorubicin had larger effects on the transporter conformation than doxorubicin. These changes are consistent with shifts in the punctate features of atomic force microscopy (AFM) images of Pgp in the absence and presence of the drugs. This information was used to develop a conformationally‐gated model for anthracycline transport by Pgp.

Support or Funding Information

NIH 1RO1CA204846‐01A1

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulation of Electron Transport by Metformin in Cardiac Protection: Role of Complex I
Authors: Mohsin Ahmed A. , Maceyka Michael , Lesnefsky Edward Joseph
Published: June 2021

Rationale

Modulation of mitochondrial complex I during reperfusion reduces cardiac injury following ischemia. Complex I exists in two structural states: active (A) and deactive (D) with transition from A→D during ischemia. Reperfusion reactivates D→A with reactive oxygen species (ROS) production. Metformin treatment may preserve the D form.

Objective

We hypothesize that maintenance of deactivation of complex I during early reperfusion will protect cardiomyocytes. The goal is to evaluate the potential of acute, high dose Metformin treatment to modulate complex I at the onset of reperfusion/reoxygenation and decrease subsequent cellular injury.

Methods

H9c2 cells underwent 6 hr. simulated ischemia and 24 hr. reoxygenation with or without metformin treatment (1 mM) at the onset of reoxygenation. Cell death, ROS and thiol modification were studied. Additional experiments were performed with or without siRNA knockdown of AMPKa2 catalytic subunit.

Results

Metformin selectively inhibited complex I oxidative phosphorylation measured at reoxygenation (Metformin: 0.34±0.1 vs control: 0.59±0.05 nmolO2/min/106cells; ±SE; n=5–10; p<0.05) with decreased total cell death following simulated ischemia and reoxygenation (Metformin: 8.3±0.2 vs. control: 14.4±0.6%; n=3–6; p<0.05). Complex II driven oxidative phosphorylation was not reduced by metformin, supporting selectively to complex I. Apoptosis assessed by annexin‐V labeling and flow cytometry was also decreased by metformin treatment. The “burst” of ROS at reoxygenation was decreased and free thiol group content was preserved by metformin treatment at reoxygenation. Metformin decreased cell death in AMPK knockdown cells.

Conclusion

Metformin blockade of complex I at the onset of reoxygenation reduces cell injury. Metformin specifically inhibited complex I at reoxygenation with less oxidative injury, in line with protection against D→A reactivation of complex I. Metformin protection was AMPK independent, supporting a complex I mechanism of protection. Acute, high dose Metformin is a potentially safe translational complex I inhibitor to treat ischemia‐reperfusion injury.

Support or Funding Information

This work was supported by the Office of Research and Development, Medical Research Service Merit Review Award (2IO1BX001355‐01A2) and Department of Veterans Affairs; National Institutes of Health NIH R21AG054975‐01

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Importance of TFEB and TFE3 in Mediating Lysosomal and Mitochondrial Adaptations
Authors: Oliveira Ashley Nicole , Memme Jonathan Matthew , Tamura Yuki , Hoood David A
Published: June 2021

Autophagy is the process through which damaged cellular components are degraded by the lysosome, and it is essential for the overall maintenance of the cell. Mitochondria are recycled through a selective form of autophagy, known as mitophagy, in which dysfunctional mitochondria are poly‐ubiquitinated, engulfed in an autophagosomal membrane and transported to the lysosome for degradation. Chronic contractile activity (CCA) alters mitophagy flux in muscle, and this coincides with the activation of lysosomal biogenesis, which precedes changes in mitochondrial content. Thus, it is proposed that the clearance of dysfunctional mitochondria through the lysosomes may play an important role in mediating mitochondrial adaptations to CCA. We sought to understand the transcriptional regulation of lysosomal biogenesis brought about by CCA, by investigating the transcriptional regulators TFEB and TFE3, and their relationship to mitochondrial content. TFEB and TFE3 were silenced using siRNA in C2C12 myotubes, and subjected to 3 days of stimulation (3hr/day) to induce CA. Following a single bout, TFEB and TFE3 were elevated in both the nuclear and cytosolic fractions, which coincided with increases in Cathepsin D, preceding the increases in VDAC seen following 3 days of stimulation. The loss of TFEB or TFE3 individually had no impact on lysosomal adaptations, since LAMP1 and Cathepsin D remained responsive to CCA, demonstrating redundancy in role of TFEB and TFE3 in mediating lysosomal biogenesis. However, in the absence of both transcription factors, CCA‐induced lysosomal adaptations were reduced, illustrating their collective importance in mediating lysosomal biogenesis. Interestingly, the absence of TFEB had no effect on mitochondrial adaptations, measured by COX I and IV protein content, however this adaptation to CCA was blunted in the absence of TFE3. These results suggest that while TFEB and TFE3 may exhibit compensatory roles in mediating lysosomal biogenesis, TFE3 alone appears to be important for the mitochondrial response to CCA. Since, lysosomal adaptations proceed in the absence of TFE3, these data suggest a more direct role of TFE3 in the regulation of mitochondria in response to chronic contractile activity.

Support or Funding Information

ANO is funded by an Ontario Graduate Scholarship (OGS). JMM is funded by Natural Science and Engineering Research (NSERC) scholarship. DAH is the holder of a Canada Research Chair in Cell Physiology with funding from NSERC and CIHR.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transcriptional and Phenotypic Characterization of Vimentin Knockout Mouse during Aging
Authors: Hu Jennifer Yuan‐Shih , Gonzalez Francisco , Davis Jennifer , Cheng Yuan , Ciesielski Mark , Ridge Karen M.
Published: June 2021

Vimentin is a type III intermediate filament (IF) that primarily found in mesenchymal cells and plays important roles in epithelial‐mesenchymal transition (EMT). IFs were originally thought to be a relatively static structure that mainly functioned as mechanical support. However, in the past decades, it has been shown that IF networks are highly dynamic structures and their roles have expanded from maintenance of cell shape and integrity to cellular migration, adhesion, and cancer metastasis. Vimentin knockout mouse was originally reported to have no overt phenotype, leading to the conclusion that vimentin IF represented an unnecessary cytoskeletal system. Interestingly, our group recently found that acute lung injury (ALI) is attenuated in mice lacking vimentin when challenged with either LPS, asbestos, or bleomycin. Moreover, these studies demonstrated vimentin to be a key player in the activation of the NACHT, LRR and PYD domains‐containing protein 3 (NLRP3) inflammasome, an intracellular protein complex that is activated in response to stimuli such as bacterial and viral pathogens, including influenza A virus. In this study, we thoroughly characterize young and aged vimentin knockout mice behaviorally using battery of neurocognitive tests and show that there are age‐dependent changes in neuromuscular, neurocognitive, and metabolic lung functions distinctively from the wild type mice. We also use RNA sequencing as an unbiased approach to study transcriptional changes and identify a significant number of differentially expressed genes between the two groups in various cell types and with aging. Additionally, histological analyses from multiple organs of these mice also suggest a crucial role of vimentin in maintaining cellular structures and functions during aging. In conclusion, our data suggests that vimentin intermediate filament plays dynamic roles in multiple organ systems and during aging.

Support or Funding Information

NHLBI R01 HL128194

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.