bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of Monocyte Age on Influenza A Virus‐induced Lung Injury
Authors: Coates Bria , Turner Margaret , Ciesielski Mark , Cheng Yuan , Ridge Karen
Published: June 2021

Rationale

Influenza A virus (IAV) causes more life‐threatening infections in healthy children than in healthy adults. In our mouse model of juvenile IAV infection, juvenile mice clear the virus at the same rate as adult mice, but develop more severe lung injury and have increased mortality compared to adult mice. We previously found that the monocytes recruited to the lungs during IAV infection exhibited a more inflammatory phenotype in juvenile mice than in adult mice. These juvenile monocytes exacerbated IAV‐induced lung injury in juvenile mice, and inhibition of their recruitment protected juvenile mice from IAV‐induced lung injury without impacting viral clearance. Based on these findings, we hypothesized that juvenile monocytes contribute to excessive inflammation and lung injury during IAV infection, and that adult monocytes could protect juvenile mice from IAV‐induced lung injury and death.

Methods

Pregnant mice were treated with busulfan (15 mg/kg i.p. day E18 and E19) to cause myelosuppression in fetal pups. Following birth, additional busulfan was given to juvenile pups (10 mg/kg i.p. day of life 1). Bone marrow derived stem cells were harvested from adult (10 week old) donor mice bearing the CD45.1 marker and injected into the myelosuppressed recipient pups (CD45.2) on day 2 of life (7.5 × 106 cells intravenously). Four weeks following transplantation, juvenile recipient mice were then infected with IAV (strain WSN, 25 PFU, intratracheally) and monitored for the development of lung injury and survival.

Results

Juvenile mice expressing juvenile CD45.2 cells had a median survival of 9 dpi. In contrast, 90% of the juvenile mice expressing adult CD45.1 were alive at 9 dpi and the median survival was >62%. Histological examination of the lungs showed decreased lung injury in IAV‐infected juvenile mice expressing adult CD45.1, as compared to control mice. Juvenile mice that recruited adult monocytes to the lungs during IAV infection had decreased lung injury compared to juvenile mice that recruited juvenile monocytes to the lungs during IAV infection.

Conclusions

Juvenile mice are more susceptible to IAV‐induced lung injury and death than adult mice. Recruitment of juvenile monocytes to the lungs during IAV infection exacerbates inflammation and worsens outcomes. Transplantation of adult monocytes into juvenile mice lessens lung injury and improves survival. This suggests that juvenile monocytes are particularly injurious in juvenile IAV infection and may have age‐related cell‐intrinsic properties that exacerbate lung injury.

Support or Funding Information

Pediatric Critical Care and Trauma Scientist Development Program (NICHD K12)American Thoracic Society/American Lung AssociationNHLBI

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Measurement and Analysis of the Mitochondrial Calcium Sequestration and Effects on the Buffering System
Authors: Strubbe Jasiel O. , Zhang Yizhu , Truong Hai , Bazil Jason N.
Published: June 2021

Calcium is a key signaling molecule found to regulate many cellular processes, including but not limited to, cellular physiology, bioenergetics, transcriptional events, signal transduction, and reactive oxygen species (ROS) production. While mitochondrial calcium dynamics have been extensively studied, it is yet unclear how calcium is sequestered and buffered in a quantitatively and mechanistic manner. Mitochondrial‐calcium homeostasis plays a crucial role in cell fate by a tightly‐regulated buffering system that maintains sub‐lethal levels of free calcium. If the calcium buffering capacity is exceeded mitochondrial dysfunction can result in excessive ROS production, initiating cell death signaling pathways, and catastrophic energy failure. In this study, we quantitatively characterized the mitochondrial calcium buffering power under a variety of experimental conditions and two calcium loading protocols. This was done with isolated cardiac mitochondria from guinea pigs using calcium‐sensitive molecular dyes. The mitochondrial calcium buffering power was quantified by monitoring the calcium flux from both outside and inside the mitochondria. Here, we report the changes in calcium buffering power caused by oxidative stress and under conditions that promote calcium uptake and retention. Furthermore, we demonstrate that the buffering power is also affected by the way calcium is loaded into the mitochondria. These data provide further insights on the mitochondrial calcium loading capacity and raises the question as to what are the components that regulate free calcium in the system.

Support or Funding Information

This research is supported by the National Heart, Lung, and Blood Institute (R00‐HL121160).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
EHD4 Regulates Prostaglandin E2 Synthesis in Renal Collecting Duct Principal Cells: Potential Implications for AQP2
Authors: Boesen Erika I. , Rahman Shamma S. , Band Hamid
Published: June 2021

Eps15 homology domain‐containing protein 4 (EHD4) regulates endocytic recycling. We previously reported that global EHD4 knockout (EHD4‐KO) mice have a higher urine flow and lower urine osmolality than wild‐type (WT) mice. Further, the diuretic phenotype in EHD4‐KO mice is associated with a reduced accumulation of aquaporin 2 (AQP2) in the apical membrane of principal cells of EHD4‐KO mice. However, the mechanism by which EHD4 regulates AQP2 localization is not known. EHD4 and AQP2 share common protein interacting partners, therefore we hypothesized that EHD4 interacts with AQP2, thereby regulating its trafficking. To test this hypothesis, we transfected mouse cortical collecting duct principal (mpkCCD) cells with a GFP‐tagged EHD4 plasmid and visualized the localization of EHD4 and AQP2. EHD4 was found to co‐localize with AQP2 in mpkCCD cells. To assess the interaction between EHD4 and AQP2, co‐immunoprecipitation of AQP2 was performed and the presence of EHD4 in the immunecomplex was measured by Western blotting. EHD4 was not detected in the immune‐complex containing AQP2, suggesting that these two proteins may not interact directly. Pointing to a potential indirect mechanism of regulation, evidence from the literature suggests that the paralog EHD1 binds to cytosolic phospholipase A2α, a key enzyme for eicosanoid synthesis. Prostaglandin E2 (PGE2) is a known negative regulator of AQP2 trafficking. Therefore, we hypothesize that EHD4 deletion increases PGE2 synthesis in principal cells, thereby contributing to the reduced membrane accumulation of AQP2. To test our hypothesis we examined urinary PGE2 excretion by female mice and found it to be significantly higher in EHD4‐KO mice (3595 ± 581 pg/day, n = 6) compared to WT mice (1042 ± 131 pg/day, n = 4; P = 0.008). To exclude a role for flow‐mediated stimulation of PGE2 synthesis, we examined the role of EHD4 in PGE2 synthesis in the mpkCCD cell line. PGE2 concentrations in the surrounding media of EHD4‐siRNA treated mpkCCD cells was significantly higher than of scrambled siRNA‐treated cells (84 ± 7 versus 53 ± 5 pg/ml respectively; n = 6; P = 0.0002). Together, these data suggest that EHD4 regulates collecting duct PGE2 production, providing a possible mechanism by which EHD4 regulates AQP2 localization and urine excretion in vivo.

Support or Funding Information

American Heart Association Grant‐In‐Aid 17GRNT33661008

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Neonatal Inflammation Impairs Multiple Pathways to Adult Respiratory Plasticity
Authors: Hocker Austin Dean , Huxtable Adrianne G.
Published: June 2021

Bacterial‐induced inflammation is a common clinical problem in neonates and acutely impairs respiratory control, but we know little about the long‐term effects of neonatal inflammation on adult respiratory control. Since neonatal inflammation blunts adult immune function, reduces adult hippocampal plasticity, and increases the vulnerability to neuropsychiatric disorders, we hypothesized that neonatal systemic inflammation would inhibit adult phrenic long‐term facilitation (pLTF), a form of respiratory neuroplasticity induced by acute intermittent hypoxia (AIH). pLTF can be induced by distinct cellular mechanisms including the Q‐pathway, induced by moderate AIH (PaO2 35–45 mmHg), and the S‐pathway, induced by severe AIH (PaO2 25–35 mmHg). Therefore, we tested the hypothesis that neonatal inflammation would undermine both the Q‐ and S‐pathway to pLTF. Postnatal day 4 (P4) pups of both sexes were injected with either lipopolysaccharide (LPS; 1 mg/kg) or sterile saline and allowed to mature to adulthood. In order to control the known effects of estrus cycle hormones on pLTF, adult females were ovariectomized 7–8 days prior and given 17‐β estradiol (40 μg/ml/kg, 3 hours) before AIH. Neonatal LPS undermined adult mAIH‐induced (Q‐pathway) pLTF in both females (−15 ± 17%, n=6, p < 0.0001) and males (14 ± 14%, n=12, p = 0.01) relative to neonatal Saline controls (females; 97 ± 24%, n=7, males; 55 ± 13%, n=7). Acute, adult treatment with a systemic anti‐inflammatory (ketoprofen, 12.5 mg/ml/kg, i.p., 3 hours) restored Q‐pathway pLTF after neonatal LPS in both females (118 ± 24%, n=4) and males (58 ± 9%, n=4) to saline controls treated with ketoprofen (P4 Saline females; 67 ± 11%, n=4, males; 54 ± 9%, n=4). Additionally, sAIH‐induced (S‐pathway) pLTF was undermined in adults after neonatal LPS in females (0 ± 17%, n=4, p < 0.001) and males (7 ± 9%, n=4, p < 0.01) compared to neonatal saline control females (86 ± 23%, n=4) and males (47 ± 17%, n=4). However, systemic ketoprofen in adults did not restore S‐pathway pLTF after neonatal inflammation in females (P4 Saline; 136 ± 11%, n=4, P4 LPS; 23 ± 15%, n=5, p < 0.0001) or males (P4 Saline; 67 ± 11%, n=4, P4 LPS; −5 ± 37%, n=4, p < 0.01). Thus, neonatal systemic inflammation has a lasting effect of respiratory plasticity by abolishing multiple pathways to adult respiratory plasticity. Furthermore, the restoration of Q‐pathway, but not S‐pathway, mediated plasticity by acute ketoprofen demonstrates different mechanisms impair adult respiratory plasticity after neonatal inflammation. Understanding how neonatal inflammation impairs the adult respiratory system will elucidate links between neonatal conditions and adult respiratory insufficiency, leading to better therapies to enhance breathing in all age groups.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Activation of Airway Afferent Vagal C‐fibers by Sphingosine‐1‐Phosphate
Authors: Patil Mayur Jayanta , Meeker Sonya , Undem Bradley
Published: June 2021

Airway inflammation leads to a release of myriad inflammatory mediators and autacoids. At the site of inflammation these molecules in theory can activate airway C‐fibers leading to the observed symptoms of reflex bronchoconstriction, mucus secretion, as well as sensations of dyspnea and urge‐to‐cough. Relatively little progress has been made to understand which among the plethora of mediators that arise during airway inflammation are most responsible for C‐fiber activation. Here we show that shingosine‐1‐phosphate, a lipid that is elevated during airway inflammatory conditions like asthma, strongly activates airway vagal C‐fibers. We employed an ex‐vivo mouse trachea/lung‐vagus nerve preparation in which action potential discharge in single afferent nerves terminating in the lung can be evaluated with extracellular recording from an electrode placed in the vagal sensory (nodose/jugular) ganglion. Drugs were delivered into the lungs directly to the receptive field. S1P (10μM) strongly stimulated, in a concentration dependent manner, 62% (n=21 out of 34 tested) of vagal C‐fibers terminating in the trachea/lungs (CV=0.63±0.04m/s), (426±71 APs, 7.2±4 Hz peak Freq). An antagonist to S1Pr3, TY 52156 (10μM) significantly blocked the stimulatory effect of S1P on S1P positive C‐fibers (214±53 APs in control vs 56±18 APs in presence of TY 52156, n=7 p=0.01). We developed an imaging technique using 2‐photon microscopy and Pirt‐GCamp6 mice to simultaneously monitor the activity of hundreds of vagal ganglion neurons in the trachea/lung‐ganglia preparation upon drug delivery into the lungs. About 40% of capsaicin (1μM) positive neurons responded to S1P (10μM). Single cell RT‐PCR revealed that ~90% of nodose ganglia neurons retrogradely labeled from the airways that express TRPV1 (denoting C‐fiber neurons) also express the S1P receptor, S1Pr3. In an RNA seq transcription analysis specifically of capsaicin‐sensitive nodose neurons we found S1Pr3 was selectively and strongly expressed. In counts per million the mRNA expression was 0, 8, 376, 3, 0 for S1Pr1, 2, 3, 4 and 5, receptors respectively. Jugular neurons expressed relatively little S1Pr receptors. We hypothesize that S1P may contribute to vagal (nodose) C‐fiber activation in airway inflammatory diseases.

Support or Funding Information

RO1 HL137807

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Probing RNA Structure and Dynamics in the HIV‐1 5′ UTR using Ensemble and Single Molecule Approaches
Authors: Musier‐Forsyth Karin , Brigham Benjamin , Kitzrow Jonathan , Reyes Joshua‐Paolo C. , Munro James
Published: June 2021

One of the most conserved and highly structured regions of the HIV‐1 genome, the 356‐nt 5′UTR, acts as a scaffold for many protein and nucleic acid binding partners and regulates vital processes including initiation of reverse transcription, genomic RNA (gRNA) dimerization and packaging. Previous studies support the presence of at least two mutually‐exclusive 5′UTR conformational states (Huthoff and Berkhout, RNA, 7, 2001; Lu et al., Science 334 (2011), but whether the 5′UTR undergoes a conformational switch during the viral lifecycle and what regulates this switch are less clear. In‐gel selective 2′‐hydroxyl acylation followed by primer extension (SHAPE) probing studies of the wild‐type (WT) 5′UTR revealed a mixed population with structural differences between monomer and dimer, consistent with a structural switch (Kenyon, JC et al, Nucleic Acids Res 41, 2013). However, these studies did not address the dynamics of the conformational change or probe possible triggers. In this work, we used single‐molecule Förster resonance energy transfer (SM‐FRET) studies to probe 5′UTR (ΔTAR/polyA) conformational dynamics and the factors that modulate it. Intra‐molecular FRET assays showed that mutations in the dimerization initiation site (DIS) and U5 region of the monomeric RNA modulated the conformational equilibrium in the expected manner, with the ΔDIS construct shifted to a high‐FRET dimerization‐competent state. Increasing Mg2+ concentration promoted the dimer‐competent conformation in the WT 5′UTR. In 10 mM Mg2+, the WT construct occupies both the high‐FRET (39%) and low‐FRET states (61%). Individual FRET traces showed that of the 841 molecules examined under these conditions, 29% transitioned between FRET states during the ~150 sec observation window. Inter‐molecular FRET observed between an immobilized donor‐labeled 5′UTR construct and excess acceptor‐labeled 5′UTR was consistent with formation of a stable extended dimer. In‐gel FRET assays were also consistent with the formation of a stable dimer. Surprisingly, tRNA‐annealing to the primer binding site of an immobilized 5′UTR promoted the monomer conformation even in the presence of high Mg2+, as observed by both SM‐FRET and native PAGE. Overall, these data are consistent with high intrinsic conformational dynamics in the 5′UTR that can be regulated by factor binding.

Support or Funding Information

National Institutes of Health

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Selenoprotein profiling in various tissues of mice fed with selenium‐deficient and high‐selenomethionine diets.
Authors: Ishii Isao , Akahoshi Noriyuki , Kamata Shotaro , Hashimoto Yuri , Hayashi Seiya , Tokoro Natsumi , Yamamoto Shingo , Shimada Ki‐ichi , Anan Yasumi
Published: June 2021

Selenium (Se) is a trace essential nonmetal element and its deficiency is known to cause myocardial necrosis leading to the heart weakening (e.g. Keshan disease). Se deficiency may contribute to Kashin‐Beck disease, resulting in atrophy, degeneration, and necrosis of cartilage tissue in the joint, or hypothyroidism including extreme fatigue, goiter, mental retardation, and miscarriages. In contrast, Se excess may cause reversible balding and brittle nails, give a garlic odor to the breath, and cause intestinal distress, weakness and slowed mental functioning. All such clinical symptoms could be attributable to dysregulated expression of twenty‐five selenoproteins in humans; mice also have the corresponding 25 selenoproteins. This study examined expression of the representative 11 selenoproteins (Gpx 1/2/3/4, TrxR1/2, Dio1, Sephs2, SelP, SelS, and SelN) in 8 different organs (brain, heart, liver, lung, kidney, pancreas, spleen, and testis) of mice fed ad libitum selenium‐deficient and high‐selenomethionine (a major dietary source from plants) diets. One week with the diets caused significant decreases and increases in Se contents of each organ, respectively, which became slightly more apparent until 4 weeks. Se deficiency in diet induced significant decreases in Gpx1/2 of liver, kidney, and pancreas; however, neither Se deficiency nor selenomethionine excess caused significant expression changes in other selenoproteins of all organs tested. There results indicate that altered organ Se contents upon Se deficiency or excess in short periods (~4 weeks) do not generally reflect selenoprotein expression levels in these organs.

Support or Funding Information

Supported by the Grants‐in‐Aid for Scientific Research (16H05107, 16K15194, 17K08287, and 25220103) from the MEXT of Japan.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein E6 in High Risk Human Papillomaviruses
Authors: Mahesh Rhea , Langat Daisy , Langat Diana , Barbara Ella , Cheng Amanda
Published: June 2021

Human Papillomavirus (HPV) refers to a group of more than one hundred HPV high risk or low risk strains associated with a variety of conditions. Over fifty strains lead to genital infections, while others influence the development of various cancers, such as cervical, skin, etc. The combination of HPV E6/E7, hTERT, and Ki67 mRNA expression levels complement each other to diagnose lesions of the aforementioned cancers, specifically dealing with the cervix. All HPV strains share a common structure: circular, double stranded DNA encoding for eight open reading frames (ORF) surrounded by a non‐enveloped capsid. ORFs function to produce the E1‐E7 proteins needed for HPV replication (early region), L1‐L2 proteins that make up the capsid (late region), and replicate and transcribe viral DNA with the help of cis components (long control region). The E6, in particular, is an oncoprotein composed of polypeptides made up of 150 amino acids and a Cys‐X‐X‐Cys motif that allow for the formation of two zinc fingers. E6 and E7 recognize a number of host proteins by hijacking their cellular domain‐motif interaction networks. In the HPV‐16 and HPV‐18 strains, the E6 protein binds its amino and carboxyl terminal regions to a leucine‐rich sequence (LxxLL) within the cellular ubiquitin ligase, E6AP. This heterodimer degrades p53, a proapoptotic tumor suppressor, although the exact mode of assembly is unknown. Complete degradation potentially leads to oncogenesis in high‐risk HPV strains, as these transformed cells have lost their ability to arrest at the G1 checkpoint within their cell cycle and perform apoptosis. Aside from the E6‐E6AP heterodimer binding directly to p53, the E6 protein has other oncogenic functions, allowing it to bind to four general classes of other proteins, providing indirect pathways to degrade p53. Binding options include proteins involved in cell polarity and motility, tumor suppressors and inducers of apoptosis, transcriptional coactivators and DNA replication/repair factors. Regarding transcription coactivators, degradation of p53 transcriptional activity is possible, as seen by the targeting of p53 coactivator p300/CBP or yADA3. Given the mechanism of E6‐E6AP complex hindering p53's from carrying out normal function, E6 is an attractive target for therapies against cervical cancers. The Olathe North High School MSOE Center for BioMolecular Modeling SMART Team used 3‐D modeling and printing technology to examine structure‐function relationships of E6.

Support or Funding Information

SMART team is a joint venture between the Milwaukee School of Engineering and the Olathe School District's 21st century program Medical Professions Academy.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluating the nuclease activity of Pseudomonas exotoxin A
Authors: Patel Nirja , Jackson Namo , Weldon John
Published: June 2021

Pseudomonas exotoxin A (PE) is a protein toxin secreted by Pseudomonas aeruginosa. The toxin is remarkably potent, and believed to be the major virulence factor of P. aeruginosa. PE inhibits protein synthesis by inactivating elongation factor 2 (EF2), the protein that advances the ribosome along the mRNA during translation. Cell with inactive EF2 are unable to synthesize proteins, which leads to apoptosis. Recent studies have indicated that, in addition to inhibition of protein synthesis, PE has also functions as a nuclease. The purpose of the experiments described here is to verify this unusual claim and characterize the nuclease activity of PE using only the catalytic fragment of the toxin. We have conducted basic time‐course and concentration assays, and have confirmed a low level of nuclease activity from PE. We are currently examining cleavage in the presence of different divalent cations (Ba2+, Sr2+, Mn2+, Co2+, Ca2+, Mg2+ and Zn2+). We have also tested a fluorescence‐based assay to observe cleavage of DNA in real‐time. Initial experiments using DNase I have been successful, and we hope to use this method to study PE in the near future.

Support or Funding Information

Research reported in this poster was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R25GM058264. Additional support for this research comes from Towson University, the Jess and Mildred Fisher College of Science and Mathematics, Graduate Studies and the Department of Biological Sciences.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploration of membrane binding of α‐synuclein through Site Based Spin Labeling
Authors: Vrabel Jarret
Published: June 2021

Neurodegenerative conditions, including Alzheimer's and Parkinson's disease, are becoming more prevalent in the aging population, and one of the main perpetrators behind these symptoms is the protein α‐synuclein (α‐syn). The cytosolic neural protein has a role in neurotransmitter transport within cells through membrane binding to intracellular vesicles. It is strongly linked to Parkinson's disease as a result of the A30P, E46K, and A53T point mutations, which are believed to cause α‐syn aggregation. These point mutations disrupt the formation of amphipathic α‐helices that directly bind to the vesicle surface. To gain further information on the membrane binding tendencies of α‐syn, each of these mutations, as well as a neutrally mutated C‐terminal tail (CTT), is being separately tested using site directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. The normally negatively charged CTT is the only region of the protein that does not directly contact the phospholipids during the membrane binding. Each of the mutant proteins' interactions with synthesized lipid vesicles will confirm the known malfunctions of the Parkinson's mutations, in addition to expanding of knowledge of the C‐terminal tail and its function in the membrane binding process. Results and analysis from the EPR spectroscopy are currently still being compiled. It is expected that each of the Parkinson's mutations and neutrally mutated CTT will decrease α‐syn membrane binding. These, or other conclusions from the analysis, can provide more information on the uncommonly studied α‐synuclein CTT and a basis on which to continue research for future undergraduate biochemistry students at Westminster College.

Support or Funding Information

Westminster College Drinko Center Undergraduate Research 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.
IMPACT OF BETA‐DYSTROGLYCAN DEFICIENCY ON EMERIN FUNCTIONS
Authors: Méndez Jesús Mauricio Ernesto Hernández , Monsiváis Wendy Lilián Gómez , González Ricardo Mondragón , Perlingeiro Rita , Mena Jaime García , Vega Bulmaro Cisneros
Published: June 2021

Beta‐dystroglycan (b‐DG) is a plasma membrane protein that belongs to the dystrophin‐associated protein complex (DAPC), the latter provides a link between the extracellular matrix and the cytoskeleton. Previously we reported that b‐DG is translocated localized to the nucleus via recognition of its nuclear localization signal (NLS), located in the yuxtamembrane domain, by importins α2/β1. Nuclear b‐DG is involved in the maintenance of nuclear envelope structure through its interaction with emerin and lamins A/C and B1. Emerin is an inner nuclear membrane protein involved in nuclear morphology, myogenesis and cell proliferation.

In this study, we analyzed the impact of β‐dystroglycan deficiency on emerin function, using C2C12 cells as model. We found that depletion of β‐DG resulted in decreased emerin levels, as shown by immunofluorescence and western blot assays with anti‐emerin antibodies. Furthermore, emerin‐mediated processes, including nuclear morphology and nucleus‐centrosome distance are altered in such cells. The analysis of emerin‐dependent nuclear signaling in β‐DG‐depleted cells is currently in progress.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Arkadia (RING finger protein 111) mediates sumoylation‐dependent stabilization of Nrf2 through K48‐linked ubiquitylation
Authors: Davis Jamaine , McIntosh Deneshia , Walters Treniqka , Arinze Ifeanyi
Published: June 2021

The transcription factor Nrf2 is a master regulator of the antioxidant defense system that serves to protect cells from oxidative damage. We previously reported that the SUMO‐targeted E3 ubiquitin ligase (STUbL), RING finger protein 4 (RNF4) accelerated the degradation rate of Nrf2 in promyelocytic leukemia‐nuclear body (PML‐NB)‐enriched fractions and decreased Nrf2‐mediated gene transcription. STUbLs contain SUMO‐interaction motifs that enable it to target substrate proteins that have been modified by SUMO resulting in ubiquitylation. These ubiquitylation events help in regulating eukaryotic cellular activity. In this study, we establish the second mammalian STUbL, Arkadia/RNF111 ubiquitylates polysumolyated Nrf2 to stabilize it, but has no effect on Nrf2‐mediated gene transcription. Interestingly, we discovered that the Arkadia‐mediated stabilization of Nrf2 occurs through Ub‐K48 linkages rather than the predicted Ub‐K63 linkages. These results suggest Arkadia‐mediated ubiquitylation of Nrf2 protects it from degradation, thereby allowing Nrf2‐dependent gene transcription. Collectively, these findings highlight a novel mechanism to positively regulate nuclear Nrf2 levels in response to oxidative stress.

Support or Funding Information

This work was supported by NIH grant SC1CA143985 and by CTSA award No. ULITR000445 from the National Center for Advancing Translational Sciences. +Supported by NIH grant # 2 S21MD000104

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phosphorylation of SLIMB by Minibrain/DYRK1a Activates SLIMB‐mediated Circadian Clock Protein Degradation
Authors: Contreras Adam , Tabuloc Chrisitne , Li Ying , Vanselow Jens , Schlosser Andreas , Chiu Joanna
Published: June 2021

SLIMB/β‐TrCP is a F‐box protein that provides substrate specificity to E3 ubiquitin ligase protein complexes, which ubiquitinate cellular proteins and target them for degradation by the 26S proteasome. Timely degradation of substrate proteins is intricately linked and critical to the regulation of numerous molecular processes, including cell cycle regulation, developmental timing, tumorigenesis, metabolism, and circadian biology. Previous work in our laboratory characterized the requirements for the interaction between SLIMB and PERIOD (PER), a key transcription factor that regulates circadian gene expression. The level of PER oscillates in a circadian manner and loss of SLIMB function disrupts PER cycling, leading to misregulation of the circadian oscillator. In contrast to the numerous studies examining the post‐translation modification (PTM) of substrate proteins to enable recognition by SLIMB, e.g. interaction of SLIMB and PER, there is a deficit of literature that investigates the PTM of F‐box proteins that modulate their function. To understand the post‐translational regulation of SLIMB function, we have identified 7 phosphorylation sites on SLIMB using mass spectrometry proteomics and characterized the function of these SLIMB PTMs in the context of the Drosophila circadian clock system.

Support or Funding Information

NIH R01 GM102225 to JCC, F31 DK104624 to AJC, and T32 GM007377

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Towards a pan‐group activator of the quorum sensing system in the common pathogen Staphylococcus epidermidis
Authors: Shen Wenqi , Tian Yang , Blackwell Helen E.
Published: June 2021

Quorum sensing (QS) is a chemical communication process that allows bacteria to regulate gene expression based on changes in population density and environmental cues, thereby enabling bacteria to coordinate their behaviors on a community‐wide scale. Among pathogens, QS is often found to play an important role in virulence. One such pathogen is the Gram‐positive bacteria Staphylococcus epidermidis. S. epidermidis is a leading cause of hospital‐acquired infections, and these infections are difficult to eradicate via traditional antibiotic therapy due to increasing antibiotic resistance and the formation of robust biofilms. The pathogenesis of S. epidermidis is mainly under the control of the accessory gene regulator (agr) QS system. agr activation is achieved by the binding of the cyclic peptide (an autoinducing peptide or AIP) signal to its cognate receptor AgrC. Synthetic compounds that target the agr system could represent valuable tools to elucidate the roles of QS and the agr system during S. epidermidis infections.

S. epidermidis can be classified into three specificity groups (I, II, or III) based on the unique AIP sequence used by each group (AIP‐I, ‐II, or ‐III). A previous study characterizing the AIP‐I signal has yielded synthetic group‐specific and pan‐group inhibitors targeting the S. epidermidis agr system. The development of pan‐group activators of the agr system is of significant interest, however, as agr activation can reduce biofilm formation by S. epidermidis. To date, no such activators have been identified. The present study was directed toward the identification of synthetic pan‐group agr activators in S. epidermidis by studying and delineating the structure‐activity relationships (SARs) of AIPs‐I, ‐II, and ‐III. Our preliminary data show that the residues at the exocyclic tail region of AIP‐II and AIP‐III are important to maintain the efficacy and potency of the native AIPs, as well as the residues that are important for receptor activation, causing a switch in certain cases from activator to inhibitor. We have also identified endocyclic residues that are important to receptor binding and activation, as well as residues that are more tolerant to change. Utilizing the findings from these systematic SARs studies for all three native AIPs, we designed a small set of AIP analogs containing features hypothesized to be important for receptor activation in each agr group, and tested these AIP analogs in all three groups using reporter strains. From this focused set of AIP analogs, we discovered the first set of pan‐group activators of S. epidermidis agr system. Current efforts are directed toward improving the potency and the efficacy of these compounds, and their application to reduce biofilm accumulation of S. epidermidis on abiotic surfaces.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In Vitro Microbiological Evaluation Of Some Metal Complexes Of 4‐Hydroxy‐2‐Oxo‐2H‐Chromene‐3‐Carbonitrile
Authors: Odai Sevdije Govori , Ibrahimi Hamide , Daci Majlinda , Haziri Arben
Published: June 2021

New complexes of 2‐Oxo‐2H‐Chromene‐3‐Carbonitrile with cobalt [Co (II)], nickel [Ni(II)], iron [Fe(III)] and chromium [Cr(III)] were synthesized. The structure of synthesized compounds were characterized by IR, 1H‐NMR and elemental analysis. The synthesized ligand and metal complexes were screened for microbiological activity against Escherichia coli Staphylococcus aureus, and Klebsiella pseumoniae. The complexes have enhanced activity compared to the 4‐Hydroxy‐2‐Oxo‐2H‐Chromene‐3‐Carbonitrile itself as ligand.

Support or Funding Information

Support for this study was provided by the Ministry for Education, Science and Technology of the Republic of Kosovo. Grant number:2‐5521‐2

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
International Research Infrastructures ‐ New research opportunities for biochemists and molecular biologists
Authors: Stechmann Bahne
Published: June 2021

In Europe, biomedical research infrastructures joined forces in the CORBEL project (www.corbel.project.eu) to offer international scientists open‐access to a wide range of state‐of‐the‐art technology platforms, resources and services in the fields of chemical biology/drug discovery (www.eu‐openscreen.eu), medical and biological imaging (www.eurobioimaging.eu), structural biology (www.structuralbiology.eu), mouse models (www.infrafrontier.eu), clinical resarch (www.ecrin.org), databases (www.elixir‐europe.org), biobanks (www.bbmri‐eric.eu), microbiology (www.mirri.org), marine biology (www.embrc.eu), systems biology (http://project.isbe.eu) and translational research (https://eatris.eu).

In the field of Chemical Biology, the academic initiative EU‐OPENSCREEN (www.eu‐openscreen.eu) integrates high‐capacity screening platforms and medicinal chemistry groups throughout Europe. Researchers from academic institutions, SMEs and industrial organisations from around the world can access the rationally selected EU‐OPENSCREEN compound collection, comprising up to 140.000 commercial and proprietary compounds collected from European chemists, to develop novel molecular tool compounds for the validation of novel biological targets, in collaboration with its partner screening platforms and medicinal chemistry groups. Molecular biologists and biochemists who have a robust and suitable assay and are interested in developing a chemical ‘tool’ compound (i.e. inhibitor or activator) for their target‐of‐interest are invited to contact us to discuss their research project. After the screening and identification of active hit compounds, researchers can collaborate with EU‐OPENSCREEN's medicinal chemistry groups during the hit‐to‐tool optimisation, use the imaging facilities of Euro‐BioImaging to characterise the biological effect of the identified compounds, or access INSTRUCT to gain structural information of the biological target.

I will highlight these research infrastructures in more detail and explain how international biochemists and molecular biologists can access and collaborate with these research infrastructures to advance their research projects.

Support or Funding Information

This project (CORBEL) receives funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 654248.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protective Effects of a Red Maple (Acer rubrum) Leaves Extract on Human Keratinocytes against H2O2‐induced Oxidative Stress
Authors: Ma Hang , Guo Hao , Liu Chang , Wan Yinsheng , Seeram Navindra P.
Published: June 2021

The red maple (Acer rubrum) species is endemic to eastern North America. It has been used as a folk medicine by the indigenous people of North America to treat dermatological ailments including skin irritation and inflammation. A series of glucitol‐core containing gallotannins (GCGs) has previously been identified from red maple aerial plant parts. Also, ginnalin A (GA), a major GCG in a proprietary phenolic‐enriched red maple leaves extract (Maplifa™), showed anti‐melanogenic effects in murine melanoma B16F10 cells by down‐regulation of tyrosinase and melanogenic gene expression as well as reduction of oxidative stress. However, the dermatological protective effects of Maplifa™ and GA on keratinocytes remain unknown. Herein, we report the protective effects of Maplifa™ and GA on hydrogen peroxide (H2O2)‐induced oxidative stress in human keratinocyte HaCaT cells. H2O2 (at 200 μM) induced cell damage of HaCaT cells and reduced cell viability by 14%. Maplifa™ (at concentrations ranging from 6.25–25 μg/mL) significantly reduced H2O2‐induced cytotoxicity in HaCaT cells and increased cell viability by 25–55%, respectively, as compared to the H2O2‐treatment group. Similarly, GA (at concentrations ranging from 6.25–50 μM) significantly increased cell viability by 34–47% respectively, as compared to the H2O2‐treatment group. In summary, Maplifa™ may have skin protective effects by reducing oxidative stress in keratinocyte HaCaT cells warranting further studies to evaluate its cosmetic applications.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of pentameric ligand‐gated ion channels by lophotoxin, a diterpenoid from coral Lophogorgia chilensis
Authors: Camacho‐Hernandez Gisela Andrea , Huber Laura , Fenical William , Taylor Palmer
Published: June 2021

Lophotoxin is a member of a cyclic diterpenoid family of toxins, devoid of nitrogen, that are isolated from the soft coral Lophogorgia. Previous work shows irreversible binding of lophotoxin to nicotinic acetylcholine receptors (nAChRs), utilizing membranes prepared from the electric organs of Torpedo californica; the inhibition is thought to be mediated by an electrophilic addition of the toxin with a hydroxyl of a conserved tyrosine (Tyr 190) of this family of receptors. According to molecular modeling, a carbon located in the epoxide moiety of the structure, may form a covalent bond that will irreversibly inhibit nAChRs. This unique family of toxins appear to bind to the agonist site of the nAChR, since binding can be prevented by reversible agonists, such as epibatidine, or competitive antagonists, such as MLA. To investigate receptor interactions of lophotoxin with neuronal nicotinic acetylcholine receptors (nAChRs) and related pentameric ligand‐gated ion channels, we employed HEK cells transfected with cDNA's encoding one of three receptor subtypes: α7‐nAChR, α4β2‐nAChR and 5HT3AR, along with a fluorescent reporter, and a receptor extracellular domain surrogate, the soluble, acetylcholine binding protein (AChBP).

We found that lophotoxin not only inhibited α7‐nAChR, α4β2‐nAChR function and AChBP ligand binding, as expected, but surprisingly also function of the serotonin gated ion channel (5HT3A). Our results suggest that these diterpinoid toxins may function not only as self‐defense agents against predators attacking branching points of the coral exoskeleton, but may also inhibit proliferation and attachment of neighboring species that could be a threat for this coral, since serotonin activation has been related to induction of germinal vesicle breakdown in bivalves and spawning in coral species. The mechanism and molecular determinants of lophotoxin interactions with pentameric ligand‐gated ion channels are still unclear, but may be resolved through X‐ray crystallographic and mass spectrometry studies with the soluble receptor surrogate, AChBP.

Support or Funding Information

CONACYT graduate fellowship to GACH

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fenugreek Saponin Improves Insulin Sensitivity in Obese Subjects ‐ A Randomized, Placebo Controlled, Pilot Study
Authors: Nair Sreejayan , Barnes Scarlette L. , Smith Derek T. , Nair Anand S.
Published: June 2021

Background

Our preclinical studies and non‐randomized clinical studies by others have demonstrated that fenugreek saponin has beneficial metabolic effects in diabetes.

Aim

The aim of this study was to evaluate whether intervention with fenugreek saponin alters body composition and cardiometabolic parameters in obese, insulin‐resistant subjects.

Trial Design

This was a prospective, double blind, placebo‐controlled, randomized, pilot clinical study.

Methods

Twenty‐eight male or female subjects with body mass index >25 Kg/m2 and satisfied one of the following 3 criteria, HbA1c >5.6%, 2 h‐post‐challenge glucose > 25 mg/dL, homeostasis model of insulin resistance (HOMA‐IR) >2.5, were randomly assigned to receive either placebo or fenugreek saponin capsules (500mg, twice daily) for 12‐weeks. Body composition (anthropometric measurements, dual energy X‐ray absorptiometry, DEXA) and clinical chemistry (serum lipids, glucose, insulin and C‐reactive protein) and enzyme‐linked immunosorbent assays (for pro‐inflammatory cytokines (tumor‐necrosis factor‐alpha) and oxidative stress (isoprostanes) were performed before and at the end of the intervention. For each parameter the difference between the pre‐ and post‐treatment values were determined and the mean of the delta value was used to determine differences between the groups.

Results

Three months intervention with fenugreek saponin had no effect on body weight, whole‐body fat mass, lean mass and android to gynoid ratio as compared to placebo. Neither did treatment with fenugreek saponin alter fasting serum lipids, insulin, glucose or HbA1c. However, the mean HOMA‐IR was significantly attenuated in the subjects receiving fenugreek saponin. Furthermore, a significant reduction in serum TNF‐alpha and isoprostane levels were observed in the treatment group.

Conclusions

Fenugreek saponin reduces insulin resistance, inflammation and oxidative stress in obese, insulin resistant subjects. Whether these effects translate to improving glucose control needs to be evaluated in larger studies in diabetic subjects.

Support or Funding Information

This work was supported by a pilot project grant from CTR‐IN which is supported by the NIH through the National Institute of General Medical Sciences #1U54GM104944‐01A1 under the Institutional Development Award (IDeA) program.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assessment of Humulus lupulus' essential oil profile and its effect on fungal infection
Authors: Walden Sean D. , Anderson C. William , Lowry Edward , Wolyniak Michael J.
Published: June 2021

Humulus lupulus, commonly known as the common hop, is a lucrative cash crop grown in large quantities across Europe and the northwest region of the United States. The mature female flowers, or cones, of H. lupulus produce a yellow, sappy substance as a defense mechanism against insect herbivory. This substance, commonly known as lupulin, contains a collection of essential oils that have signature strong flavors and aromatic qualities that give hops their commercial value in brewing. Beer and mead, the products of hops‐mediated brewing, obtain their unique flavors through the different combinations of oils produced by different varieties of H. lupulus.

While the anti‐herbivory properties of H. lupulus essential oils are well‐established, relatively less is known about their role in suppressing the growth of pathogenic fungi. Indeed, H. lupulus populations have been plagued by Downy Mildew (Pseudoperonospora humuli) and Powdery Mildew (Podosphaera macularis). These pathogens can infect entire commercial crops in a short amount of time and can cost commercial producers a significant amount of money if crops become significantly infected. Coincidentally, the climate which favors growth of P. humuli and P. macularis is also the climate in which H. lupulus grow best. This fact underscores the importance of better understanding how to control the growth of these fungi with respect to the future of H. lupulus agriculture. In a recent study, essential oils from thyme (Thymus vulgaris L.) have been shown to have anti‐fungal qualities (Zambonelli 1996). Thus, the essential oils produced by H. lupulus could similarly be effective in preventing infection by these pathogens.

With the goal of better understanding the role of essential oils and their distinct profiles in the many varieties of H. lupulus, five common varieties were profiled using Gas Chromatography‐Mass Spectroscopy (GC‐MS). This technique provides a holistic look at the different oils produced in different varieties of H. lupulus. Preliminary work has suggested that the most prominent essential oils present in all tested varieties includes humulene, beta‐myrcene, beta‐caryophyllene, and lupulon. Beta‐caryophyllene is especially interesting in these results based on its previously determined role as an anti‐fungal agent. Humulene is also interesting due to it being an isomer of beta‐caryophyllene. There are some less abundant oils in the profiles that are unique to distinct varieties and which are currently being analyzed through a measure of zone of inhibition following inoculation with individual essential oils on impregnated disks exposed to fungal cultures on potato dextrose agar (PDA) medium.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.