bluuebunny/crossref_metadata_2025 huggingface dataset:
Antioxidant Activity and Phenolic Content of Chinese Balsam (Impatiens chinensis)
Authors: Oldenburg Kimberly , Henning Shelby , Soendergaard Mette
Published: April 2020

Plants of the Impatiens genus includes more than 1,000 species. Several of the species are used as medicinal plants and are known to contain the anti‐inflammatory 2‐methoxy‐1,4‐naphthoquinone. Herbal remedies are used to treat insect bites, rashes, and poison ivy contact dermatitis, as well as management of anxiety and stress. Chinese Balsam (Impatiens chinensis) is found in North‐East India, where it is used medicinally for pain relief, promoting blood circulation, and treating urinary infections. However, very little research has been conducted to analyze the phytochemistry of I. chinensis. Here, is described the first report of antioxidant capacity and phenolic content of the seeds, leaves, stems, roots and flowers of I. chinensis.

Impatiens chinensis plants were separated into seeds, leaves, stems, roots and flowers, freeze‐dried, and ground into a powder. For antioxidant assays, ground plant powder was extracted using 1% HCl, 90% aqueous methanol for 2 h under shaking. The 2,2‐diphenly‐1picryl‐hydrazyl‐hydrate (DPPH) and (ABTS) assays were used to determine the antioxidant capacity of each extract. Briefly, 10 μL extract was incubated with 10 μM DPPH for 10 min, after which the absorbance was measured spectrophotometrically at 517 nm. For the ABTS assay, 10 μL extract was incubated with 3.5 mM ABTS radical for 30 s. The absorbance at 734 nm was then measured spectrophotometrically. Results from both assays were compared to a trolox standard and calculated as 6‐hydroxy‐2,5,7,8‐tetramethylchroman‐2‐carboxylic acid (trolox) equivalents (TE; μmol/g dry weight). Results from the ABTS assay showed antioxidant activities of 189.5±26.7, 7462±286.3, 195.3±31.6, 85.03±19.7, and 484.1±29.3 TE (mean±SEM) for stems, flowers, leaves, roots, and seeds, respectively. In correlation, the DPPH assay revealed the same pattern of radical scavenging ability in the various tissues, with the flowers exhibiting the highest amount of antioxidant capacity. The stems, flowers, leaves, roots, and seeds exhibited 150.1±6.128, 910.8±31.62, 188.8±10.72, 118.2±13.27, and 486.9±17.81 TE (mean±SEM), respectively.

The phenolic content of I. chinensis extracts was determined using the Folin‐Ciocalteu assay. 20 μL extract was incubated with 2N 10 μL extract reagent, ddH2O, and 12.56% sodium carbonate. After 30 min, the absorbance at 750 nm was measured spectrophotometrically. Results were compared to gallic acid as a standard, and the phenolic content was calculated as gallic acid equivalents (GAE; μmol/g dry weight). The phenolic content was 1127±42.6, 5046±151.0, 1206±49.3, 662.2±24.5, and 1270±70.5 GAE (mean±SEM).
The Structural Characterization of AtLaRP6a C‐Terminal Domain
Authors: Hackler Ezra Lawrence Clark , Lewis Karen
Published: April 2020

The La related proteins (LaRPs) are a superfamily of RNA binding proteins characterized by a conserved sequence called the La Module. This domain is composed of two distinct regions, the La Motif (LAM) and an RNA recognition motif (RRM), which form a functional RNA binding unit. The LaRP6 subfamily is characterized by a putative protein‐protein interaction motif in the C‐terminus, called the LAM‐ and S1 associated (LSA) motif. Interestingly, plants express three paralogs of LaRP6 (A, B, and C). We have recombinantly expressed and purified the LaRP6A protein from the plant model organism Arabidopsis thaliana, to compare the plant LaRP6A to the LaRP6 proteins of vertebrate organisms for the purpose of probing differences in structure and function. Previous work in our lab suggested that the C‐terminal domain of AtLaRP6A is intrinsically disordered. In this study, we have recombinantly expressed and purified only the C‐terminal domain. Biochemical and biophysical analyses, including analytical size exclusion chromatography, limited proteolysis, and circular dichroism, all support the prediction that the CTD is an intrinsically disordered region. This work provides a foundation to further study the role of the CTD in regulatory protein‐protein interactions to better understand the biological role for LaRP6.
Increased Toluene Exposure and Toxicity Effects Results in Low Fly Fecundity and Offspring Development in Female Drosophila melanogaster
Authors: Ullah Emaad , Arredondo Dalia , Moreno Angela , Campanas Mary , Lopez Francisco , Kay Gabrielle , Nguyen Andrew , Rosell Rosemarie
Published: April 2020

Toluene is a volatile hydrocarbon found in industrial settings and in household products. It is classified as noncancerous but air‐toxic, and chronic exposure to toluene can have negative effects on human neurological function, organ function, fetal development, and can cause hormonal changes. Our purpose is to further investigate the effects of toluene on a population of the model organism, Drosophila melanogaster. Since humans and D. melanogaster show many similarities in body systems structure and function, we found that exposure to toluene will affect the reproductive system and fecundity in female fruit flies. We hypothesize that by now exposing males and tracking through new methods of observation that toluene exposure will also affect their nervous and reproductive systems. To observe these effects, a continuous flow system that delivers known concentrations of gaseous toluene to populations of D. melanogaster was developed. We have interpolated the lethal concentration of gaseous toluene that kills 50% of the population (LE50) to be between 1000 and 1200 ppm. Female adults exposed at concentrations just above and below the LE50 were mated with unexposed male fruit flies and the resulting offspring were counted at egg, larval, and pupal stages. These data showed that as toluene concentration increased there appears to be a decrease in fly fecundity and offspring survivability, indicating that the effects of toluene are dose dependent and have detrimental effects on fly development. Future studies include determining the correlation between toluene exposure to fly offspring morphology and larval development. As well as exposing male fruit flies to toluene and measuring the reproductive development from this exposed progeny. Additionally, differential transcriptional analyses will be conducted, to determine if phenotypic changes are seen during development have a genetic basis.
ECG Assessment of Cardiac Function Before, During, and After a 1km Swim Performed in Normal and Cold‐Water
Authors: Ahlers Kristina , Clementson Samantha , Lentz Mason , Fell Megan , Frie Kathryn , Harrison Michael , Johnson Bruce , Wilson Ted
Published: April 2020

Cold‐water swimming is a recreational activity that may become a future Winter Olympic event. However, unintentional cold‐water exposure can lead to arrhythmias, changes in electrocardiogram (ECG) appearance, and death. This study sought to characterize cardiac responses to recreational one‐kilometer swims performed in normal and cold‐water. Competitive swimmers (2 male 2 female; age 44±17 years; BMI 24±2) completed a one‐kilometer swim (22 laps × 75‐foot lengths) under free‐living indoor (25.5°C water and 25.0°C air) and cold outdoor (10.9°C water and 6.5°C) conditions. Continuous ECGs were obtained with a waterproof recording system (Actiwave, CamNtech Inc., Boerne, TX). ECG PR‐, QT‐, and RR‐intervals (sec), ventricular diastole (sec), and heart rate (beats/minute) were measured 8.7±1.7 minutes before the swim, then 0.6±0.2, 3.0±0.0 and 6.4±2.1 minutes after the swim. Data (mean±SD) was analyzed with repeated measures ANOVA with significance if P<0.05. Swimmers completed the indoor and outdoor swim events in 20.7±1.7 and 21.8±1.1 minutes, respectively. For the indoor swim, PR‐interval (sec) at −8.7, +0.6, +3, and +6.4 minutes was 0.12±0.01, 0.12±0.01, 0.11±0.01, and 0.12±0.01, and PR‐interval for the outdoor swim at −8.7, +0.6, +3.0 and +6.4 minutes was 0.13±0.01, 0.13±0.01, 0.13±0.01, and 0.14±0.01 (sec). PR‐interval was significantly longer at +6.4 minutes post cold‐water swim. For the indoor swim, ventricular diastole (sec) at −8.7, +0.6, +3, and +6.4 minutes was 0.37±0.06, 0.22±0.04, 0.30±0.01, and 0.32±0.05 (sec), and for the outdoor swim at −8.7, +0.6, +3.0 and +6.4 minutes ventricular diastole was 0.35±0.08, 0.19±0.02, 0.30±0.04, and 0.24±0.06 (sec). Ventricular diastole was significantly shorter at +6.4 minutes post cold‐water swim. No significant changes were observed for QT‐interval, RR‐interval, and heart rate between swim conditions. These parameters were also assessed for the ECG recordings collected during the 20.7±1.7‐minute normal and 21.8±1.1‐minute cold‐water swims to characterize the physiological response during swim exercise. This study of normal and cold‐water one‐kilometer recreational swimmers determined that a waterproof ECG could generate a continuous record of changes in cardiac function. During cold‐water swim recovery (+6.4 minutes) PR interval was lengthened and ventricular diastole was shortened suggesting an influence of temperature on cardiac function.
Regulatory Role of Ceramide/mTOR Signaling in Exosome Secretion from Smooth Muscle Cells during Arterial Stiffening and Medial Calcification
Authors: Bhat Owais M. , Yuan Xinxu , Li Guangbi , Camus Sarah , Kukreja Rakesh C. , Gulbins Erich , Li Pin-Lan
Published: April 2020

Increased secretion of exosomes from arterial smooth muscle cells (SMCs) has been shown to contribute to arterial medial calcification (AMC) associated with accumulation of calcium deposits in the arterial wall and osteoblastic differentiation of SMCs. In this study, we further explored the molecular mechanisms increasing exosome secretion during AMC, focusing on lysosomal‐ceramide/mTOR (mammalian target of rapamycin) signaling in the regulation of lysosome trafficking and exosome release. Smooth muscle‐specific lysosomal acid ceramidase (Ac) gene knock out (Asah1fl/fl/SMcre) mice were treated with a high dose of Vit D (500,000 IU/kg/day) for 4 days. By Alizarin Red S and Von Kossa staining, we observed significantly increased aortic medial calcification with augmented expression of osteogenic markers like osteopontin and RUNX2 in Asah1fl/fl/SMcre mice compared to their wild type littermates, which was markedly attenuated by pretreatment of these mice with Torin‐1, an mTOR inhibitor. Confocal microscopy showed Asah1fl/fl/SMcre mice had markedly increased colocalization of mTORC1 with Lamp‐1(lysosome marker), but decreased colocalization of VPS16 (a multivesicular bodies (MVBs) marker) with Lamp‐1, suggesting mTOR activation and reduced MVBs interaction with lysosomes. However, mTOR inhibition by Torin‐1 significantly reduced the colocalization of mTOR vs Lamp‐1 which inturn increased lysosome‐MVBs interaction. Torin‐1 was also found to reduce accumulation of CD63 and annexin 2 (AnX2) (exosome markers) in the coronary arterial wall of Asah1fl/fl/SMcre mice compared to wild type littermates. Using CASMCs, we observed that Ac gene deletion led to peripheral distribution of lysosomes and increased exosome secretion, which was blocked by Torin‐1. Functionally, Torin‐1 was found to significantly reduce arterial stiffening Asah1fl/fl/SMcre mice as shown by restoration from increased pulse wave velocity (PWV). Altogether, these results suggest that lysosomal ceramide‐mTOR signaling is critical for the control of lysosome trafficking, exosome secretion and arterial stiffening or osteogenesis.

Support or Funding Information

Supported by NIH grants HL057244, HL075316 and DK120491
Development of Picomolar Affinity Antagonists and Long‐acting Agonists for the Adrenomedullin and CGRP Receptors Using Combinatorial Peptide Library and Structure‐guided Design Approaches
Authors: Pioszak Augen A. , Booe Jason M. , Warner Margaret L.
Published: April 2020

Adrenomedullin (AM) and calcitonin gene‐related peptide (CGRP) have diverse functions in the cardiovascular, lymphatic, and nervous systems including regulation of vascular tone, cardioprotection, and pain transmission. These peptides signal through three heterodimeric receptors comprising the class B GPCR, the calcitonin receptor‐like receptor (CLR), and one of three receptor activity‐modifying proteins (RAMP1‐3) that determine ligand selectivity. CLR‐RAMP complexes are proven or promising drug targets for several diseases, however, the moderate binding affinities and short plasma half‐lives of AM and CGRP limit their utility as therapeutics. Here, we used synthetic peptide combinatorial library and rational structure‐guided design approaches to develop AM and CGRP variant peptides with dramatically enhanced receptor affinities. The binding of the variants to purified RAMP‐CLR extracellular domain (ECD) complexes was characterized and a high‐resolution crystal structure of an AM variant bound to RAMP2‐CLR ECD explained its enhanced affinity. Incorporation of the variants into different length AM and CGRP scaffolds yielded picomolar affinity antagonists or agonists that exhibited substantially prolonged cAMP signaling after ligand washout. These activities were demonstrated in a model cell line transiently expressing the receptors and in primary human cells or immortalized cell lines that naturally express the receptors. This work provides a suite of AM and CGRP variants that will be valuable pharmacological tools and may have promise as peptide therapeutics with long receptor residence times. In addition, the properties of the variants support a mechanism for RAMP function in which direct RAMP‐peptide contacts and allosteric modulation of CLR cooperate to determine ligand selectivity.

Support or Funding Information

This work was supported by grants NIH R01GM104251 and OCAST HR16‐005 (AAP) and AHA predoctoral fellowship 18PRE33990152 (JMB).
A Study on Morphometric Features of Trachea and Larynx
Authors: Alimohammadi Majid , Doroudi Majid , Ebrahimi Azarnoush , Zilabi Golsan
Published: April 2020

Certain data such as weight and dimensions of structures are important in dosimetry for treating cancer patients using radiotherapy.

The objective of this study is to determine the morphometric features of trachea and larynx and discuss the possible correlation between the height and weight of these structures with other parameters such as height, weight and age of patients. 29 samples were measured using measuring tape and digital scale. The results of measurements showed significant correlation between the age of individuals and the weight and length of trachea and larynx. However, no significant correlation was found between the length and weight of trachea or larynx with either gender, height or weight of individuals.
Characterization of a Splice Variant of Perilipin 5
Authors: Huston Matthew R. , Tansey John T.
Published: April 2020

Lipid storage provides energy stores for the body in times of increased oxidative requirements, such as fasting or exercise, but when the balance of lipid storage and mobilization is disrupted human health suffers. Implications for increased lipid storage include risk factors for developing type II diabetes mellitus and non‐alcoholic fatty liver disease. The perilipin family of proteins associate with the surface of lipid droplets and are essential to regulation of stored triacylglycerols. Perilipin 5 functions to maintain a balance between the storage of triacylglycerols in lipid droplets or their mobilization when energetically required. Several members of the perilipin gene family exhibit differential splicing, most notably perilipin 1 but reports exist of alternative messages for shortened forms of perilipins 2 and 3. We have identified a shortened form of perilipin 5 we have termed perilipin 5b. Western blots indicate that the protein, of approximately 35 kDa, is present in C2C12 cultured myoblasts and in heart and liver tissue from fed and fasted mice. Analysis of murine genomic DNA sequences reveals stop codons found in the introns between exons 6 and 7 and 7 and 8, either of which could give rise to a protein of approximately 35 kDa, indicating the likelihood of the observed protein being a splice variant, rather than a post‐translationally modified protein. Using RT‐PCR we have identified multiple amplicons suggesting that perilipin‐5b contains a read through of intron 6 of the murine perilipin 5 gene. Analysis of the amino acid sequence of perilipin 5b indicates that this truncation would lack the reported C‐terminal domain essential for interactions with ATGL and CGI‐58 but would retain the PKA site found at Ser155. The function of perilipin 5b is yet unknown, but the fact that it is found primarily in heart tissue seems to suggest that it plays a role in the oxidation of the lipid droplet. These data are consistent with what is currently known about Perilipin‐5 and the other PAT family proteins. Determination of perilipin 5b function will give a more accurate picture of the overall mechanism of lipid storage and metabolism.
Orphan sensory receptors are expressed in hepatocytes
Authors: Shepard Blythe D.
Published: April 2020

Sensory receptors, including olfactory receptors (ORs), taste receptors (TAS), and orphan G‐protein coupled receptors (GPRs) have recently been found in a variety of non‐sensory tissues where they have important physiological functions. As G protein‐coupled receptors (GPCRs), these proteins serve as chemosensors by interpreting and responding to chemical cues in their native environment (i.e. blood, urine) and can be activated by naturally occurring metabolites. We reasoned that the liver, the largest metabolic organ in the body, is primed to take advantage of some of these sensory receptors in order to regulate its internal environment. To identify the complete complement of sensory receptors expressed in murine liver, we performed an unbiased screen of whole liver using TaqMan qPCR array cards. This screen identified a total of 17 ORs, 10 TAS, 3 opsins, and 57 GPRs – with 7 of the GPRs expressed at levels equal to well‐characterized hepatic GPCRs such as glucagon receptor. In order to understand the functions of these sensory receptors, it is imperative to determine where they localize and their ligand profile. Due to the lack of reliable antibodies, we turned to RNAScope to localize mRNA transcript expression within the liver. Using this technique, we were able to confirm that murine Tas2r108, Olfr57, and Gpr137 are all expressed in hepatocytes, the metabolically active epithelial cells of the liver. With confirmation of expression, all three of these hepatic receptors were cloned from both male and female livers and expressed into heterologous cells for ligand screening. While Tas2r108 has known ligands – bitter tastants that have anti‐bacterial functions – both Olfr57 and Gpr137 remain orphan receptors. This is due, in part, to the inability to achieve functional expression of these receptors in vitro. Of note, Gpr137 has multiple splice variants and co‐expression of the canonical full‐length GPCR form along with a truncated variant increases surface expression of this 7‐transmembrane domain receptor. Ongoing studies are aimed at determining the role that Gpr137 plays in hepatic protein trafficking. Surface expression conditions were also achieved for Olfr57 with the assistance of a cleavable LUCY tag allowing for large‐scale ligand screening using a cAMP‐driven dual luciferase reporter assay. Using this platform, we determined that Olfr57 responds to terpenes including camphor and fenchol. These volatile organic compounds are known fungal metabolites, opening up the possibility that the liver is utilizing ORs to detect fungal growth. Efforts are currently underway to test this hypothesis and to establish the functional relevance for these chemosensors in hepatocytes.

Support or Funding Information

NIDDK
Novel roles of Atrogin‐1 in cardiac disease, lipid metabolism and bone microstructure
Authors: Klüppel Michael , Suryadevara Vidyani , Willis Monte S.
Published: April 2020

Ubiquitination plays a critical role in controlling protein turnover via proteasomal degradation, altering subcellular localization, and regulating protein activity. Functional role(s) for the Atrogin‐1 (MAFbx) ubiquitin ligase in regulating transcription factors have been found in skeletal muscle and the heart, but not in other organs. Within the myocyte, Atrogin‐1 localizes to the sarcomere and nucleus. We have previously reported that Atrogin‐1 is a critical regulator of pathologic and physiologic cardiac hypertrophy in vivo, involving specific interaction and ubiquitination of transcription factors central the signaling pathways driving these hypertrophic responses, including FOXO1/3. Recent studies have linked cardiomyocyte Atrogin‐1 to the regulation of the extracellular matrix. With evidence that cardiomyocyte Atrogin‐1 can affect cells other than myocytes, we analyzed Atrogin‐1−/− mice for their effects on metabolism and bone. DEXA whole body analysis of Atrogin‐1−/− mice revealed significantly decreased fat mass (15.6% vs. 35.8% fat, p<0.05) with a corresponding increase in lean mass (84.4% vs. 64.2%, p<0.05) at the age of 11–15 months, but not at 5 months, when compared to strain‐ and age‐matched wildtype (WT) control mice. Since the Atrogin‐1−/− mice weighed significantly less than WT mice (29±3 vs. 40±2 g, p<0.05), the net fat loss was 4.6 g (vs. 14.3 g in age‐matched WT) and the lean body mass identical between groups (25.7 vs. 24.6 g). Together these findings suggest Atrogin‐1’s role in regulating fat metabolism. Analysis of bone microarchitecture and mechanical properties of adult Atrogin‐1−/− mice was undertaken at 17–21 weeks of age, a time point where no phenotype has been previously observed in skeletal muscle or heart. Femurs were dissected and subsequently assessed by μCT (SkyScan 1172) for bone microarchitecture in the distal femur metaphysis (1 mm) and mid‐diaphysis region and challenged by a three‐point bending test to assess their material and structural properties. No differences in cortical or trabecular bone microarchitecture were identified and nor were any changes in mechanical properties. Together, these findings illustrate novel biological roles of Atrogin‐1 in regulating systemic fat metabolism, possibly involving crosstalk between skeletal muscle/heart and systemic fat metabolism. Our analysis provides an essential framework for the potential therapeutic targeting of ubiquitin ligases like Atrogin‐1 in the context of striated muscle and metabolic disease.

Support or Funding Information

Support: Lilly Foundation/Indiana Center for Musculoskeletal Health/ IU School of Medicine, Physician Scientist Initiative, Scientific Research Initiative
Chemerin Increases Contractility of the Uterine Artery and Leads to Placentomegalia
Authors: Justina Vanessa Dela , Priviero Fernanda , Lima Victor Vitorino , Giachini Fernanda Regina , Webb R. Clinton
Published: April 2020

Chemerin is an adipokine released from several tissues (mostly adipocytes but also placenta, ovaries and liver) which participates in the regulation of adipocyte development, inflammatory processes, and metabolic functions. Chemerin is gaining attention in gestation and it has been reported to be augmented in preeclampsia. This adipokine plays a physiological and protective role during early pregnancy; however, late complications in pregnancy may be observed when chemerin levels remain augmented. During pregnancy, maternal uterine artery (UA) blood flow is critical to maintaining the intrauterine environment, permitting normal placental function, and supporting fetal growth. Chemerin has been shown to contract many vascular beds, however, its role on the UA remains to be elucidated. This study tests the hypothesis that chemerin leads to increased contraction of UA contributing to the development of preeclampsia‐like symptoms and leading to gestational disorders. Pregnant (P) and non‐pregnant (NP) Sprague Dawley rats (200–250g) were injected daily (i.p.) with vehicle (saline) or chemerin (16 μg/rat;). On gestational day 20, or corresponding day for NP rats, animals were euthanized and placentas and fetuses weighted. UA was removed, cleaned and mounted in wire myograph. Concentration response‐curves to acetylcholine (ACh) and seretonin [5‐HT (1 nM to 100 μM); in the presence or absence of ERK1/2 inhibitor, PD98059] were performed in UA. Treatment with chemerin decreases the relaxation to ACh in both P [Emax 69.77 ± 4.90 vs. 95.50 ± 3.10] and NP [Emax 74.56 ± 4.52 vs. 96.04 ± 5.75] animals compared to respective controls. Increased sensitivity to 5‐HT was observed in the UAs of chemerin‐treated P [pD2 7.05 ± 0.05 vs. 6.60 ± 0.16] and NP [pD2 6.57 ± 0.07 vs. 6.14 ± 0.14] rats compared to vehicle rats, which was abolished by incubation with PD98059. Further, chemerin treatment on P animals resulted in increased placental weight [(g) 0.50 ± 0.016 vs. 0.43 ± 0.004] with no changes in fetal weight. We conclude that chemerin treatment impairs UA contractility and relaxation leading to placental disorder. These changes in UA function may be a predictive for elevate blood pressure in preeclampsia.

Support or Funding Information

NIH 13604
LXR AGONISM POTENTIATES SORAFENIB ACTIVITY IN HCC BY INDUCING METABOLIC STRESS
Authors: Preziosi Morgan Elizabeth , Zahm Adam , Kaestner Klaus , Wangensteen Kirk
Published: April 2020

Hepatocellular carcinoma (HCC) treatments have low efficacy, as FDA‐approved drugs including sorafenib extend patient survival by three months. We sought to identify novel druggable targets for use in combination with sorafenib to increase its efficacy. We implemented a genetic screening paradigm using an overexpression library of 43 genes. We administered this library in the context of liver injury and treated mice with vehicle or sorafenib and determined the genetic drivers of each tumor. Mice injected with the screening library developed HCC clones containing MYC cDNA plus various other cDNAs. Treatment with sorafenib resulted in sorafenib‐resistant HCCs that were significantly depleted in NR1H3 cDNA, encoding Liver X receptor (LXR), suggesting that LXR activation is incompatible with tumor growth during sorafenib treatment in vivo. Combination treatment using sorafenib and LXR agonist GW3965 in multiple HCC cell lines and a primary cell line led to enhanced cell death as compared to monotherapy, due to reduced expression levels of cell cycle regulators and increased expression of genes linked to apoptosis. Pathway analysis revealed alterations in metabolic pathways, and we observed upregulation of gluconeogenic regulators and downregulation of glycolytic regulators in sorafenib and combination treatment, and increased regulation of fatty acid synthesis genes in GW3965 and combination treatment. When gluconeogenesis and fatty acid synthesis are reduced using siRNA, cells are resistant to combination therapy. We propose that sorafenib upregulates glucose synthesis and downregulates glycolysis, while GW3965 upregulates fatty acid synthesis. In combination, sorafenib and GW3965 deplete cellular resources required for ATP production, subsequently leading to apoptosis.
Cap‐independent translation of a subset of cellular mRNAs is driven by the direct recruitment of eIF4G or DAP5 to the 5′ UTR of these mRNAs
Authors: Haizel Solomon A. , Bhardwaj Usha , Gonzalez Ruben L. , Goss Dixie J.
Published: April 2020

Apoptosis, hypoxia, and other stress conditions inhibit canonical, cap‐dependent translation by triggering overexpression of 4E‐binding protein (4E‐BP) and increasing 4E‐BP mediated sequestration of eIF4E. Under these conditions a subset of cellular mRNAs encoding proteins with important roles in human health and disease, including HIF‐1α, FGF‐9 and p53, are known to translate in a cap‐independent manner. Interestingly, eukaryotic initiation factor 4G (eIF4G) and/or DAP5 are also overexpressed under these stress conditions, suggesting eIF4G/DAP5 might play a role in the cap‐independent translation of cellular mRNAs during stress. Despite their physiological importance, however, the molecular mechanisms underlying cap‐independent initiation of this subset of cellular mRNAs remain unknown. To address this gap in our understanding, we have used fluorescence anisotropy‐based binding assays developed in our laboratories to demonstrate that two N‐terminal truncated forms of eIF4G (i) eIF4G 682‐1599 that cannot interact with eIF4E and (ii) eIF4G 557‐1599 that contains the eIF4E binding domain and or DAP5 can directly bind specifically and differentially to structural elements that are found in the 5′ UTRs of this subset of cellular mRNAs. We also report that the eIF4E binding domain on eIF4G 557‐1599 mutant increases the selectivity among the mRNAs even in the absence of eIF4E. Using a luciferase‐based gene expression reporter assay, we further demonstrate that these same structural elements can promote translation initiation in an eIF4GI or DAP5‐dependent manner in a nuclease‐treated rabbit reticulocyte lysate depleted of eIF4GI or DAP5. Collectively, our studies are providing quantitative binding data and in vitro translation data that are allowing us to elucidate how a subset of cellular mRNAs switch from cap‐dependent to cap‐independent modes of translation initiation; how the structural elements in these mRNAs recruit eIF4GI and/or DAP5 during cap‐independent translation initiation and how eIF4GI and/or DAP5 enables the subsequent assembly of the translation initiation complex.

Support or Funding Information

National Institutes of Health [NIH R01 GM 084288 to R.L.G]; National Center for Advancing Translational Sciences [1UL1TR002384‐01 Seed Project to D.J.G] and National Institutes of Health [NIH R01 GM 128239 to D.J.G]
Gene delivery to mid‐cervical dorsal root ganglia using AAV9
Authors: Benevides Ethan S. , Sunshine Michael D. , Fuller David D.
Published: April 2020

Delivery of viruses (e.g., adeno‐associated virus, AAV) or small molecule tracers (e.g., cholera toxin) to the pleural space between the lung and chest wall (i.e., “intrapleural delivery”) can effectively target phrenic motor neurons (see Mantilla et al., 2009, Journal of Neuroscience Methods). In ongoing experiments we are evaluating the effectiveness of intrapleural delivery of AAV serotype 9 (AAV9) for driving transgene expression in diaphragm sensory neurons in the cervical dorsal root ganglia (DRG) in C57/bl6 mice. Initial experiments used AAV9‐CBA‐GFP (titer: 1.42–8.11×1012 vg/ml) with relatively low total volume (6 μl of virus diluted in 6 μl of 1x PBS) for bilateral intrapleural injection; this approach failed to produce noticeable DRG transgene expression. We next tried larger unilateral doses (33 μl virus) diluted into 1x PBS to make a larger total volume (400 μl); this approach produced cervical DRG GFP expression, but positive DRG somata were sparse. Ultimately we utilized a higher titer AAV9‐CBA‐GFP (1.48×1013) at several doses: 33 μl, 66 μl, 100 μl, or 200 μl (n=2–3 per group). All doses were diluted up to 400 μl total volume in 1x PBS. After 6–8 weeks mice were perfused with 4% paraformaldehyde (PFA) and the central nervous system was harvested, post‐fixed in 4% PFA, and cryo‐protected in 30% sucrose. Brains and spinal cords were cryo‐sectioned (40 μm) and imaged using a fluorescence microscope for GFP expression. All the doses showed unequivocally positive GFP expression in afferent fibers in the mid‐cervical dorsal roots as well as DRG somata. Initial qualitative evaluation indicates a dose‐dependent increase in total cervical DRG labeling progressing from 33–100 μl, with no additional labeling at 200 μl. Interestingly, GFP expression was detected in very few phrenic motoneurons, even at the high doses. Our preliminary conclusion is that intrapleural injection of relatively large volumes of high titer AAV9 can drive transgene expression in mid‐cervical DRG neurons.

Support or Funding Information

Support: SPARC OT2 OD023854, F31 HL145831‐01 (MDS)
Influenza versus Host: The Role of Novel Copper‐Related Host Factors in Antiviral Immunity
Authors: Dagdag Ralf P. , Burkhead Jason L. , Bortz Eric
Published: April 2020

Influenza A virus is a respiratory orthomyxovirus that causes 3–5 million cases of severe illness and 250,000–500,000 deaths annually in humans. The NS1 protein antagonizes RIG‐I and IRF3‐induced IFN‐β responses during an influenza infection. Some influenza strains also contain nucleoproteins (NP) variants that impart resistance to the antiviral protein Mx. To better understand roles of NP in antagonizing interferon responses, NP plasmids from H1N1, H5N1, or H7N9 influenza subtypes were transfected into A549 human lung adenocarcinoma and HEK293T IFN‐β luciferase reporter cells. The viral RNA mimic poly‐I:C was used to induce a type‐I IFN response. Quantitative measures of normalized luciferase values (RLUs) reveal that viral NPs significant decrease the expression of both IFN‐β and the antiviral gene ISG56, indicating antagonism of host innate immune responses by NP.

On the host side, copper handling machinery has been recently recognized as regulating influenza infection. RNA interference experiments showed that copper transporter ATP7A, a protein that imports [Cu1+] into trans‐Golgi derived compartments, is required for influenza viral RNA and protein syntheses. The trafficking‐regulatory Commander complex includes the 10‐member COMMD protein family, first identified in regulation of copper export. Preliminary experiments reveal that a type I IFN response induced by both low and high molecular weight variants of poly‐I:C in A549 cells also results in dose‐dependent upregulation of COMMD5 (13‐fold) and COMMD7 (24‐fold), but not COMMD1. Viral mixed infections using A/WSN/33 (H1N1) and A/PR/8/34 (H1N1) result in the upregulation of COMMD5 and no significant change in COMMD1 or COMMD5, which may be a result of immunosuppression by the viral proteins NS1 and NP. While simultaneous knockdown of COMMD5 and COMMD7 did not enhance influenza infection of A549 cells, knockdown of the copper‐binding protein ATOX1 licensed viral replication in a limiting dilution assay. Taken together, these results suggest a previously unrecognized interplay between viral NP, antiviral proteins and copper‐binding proteins to regulate influenza A virus infection in mammals, providing novel targets for antiviral therapy.

Support or Funding Information

Alaska INBRE
Functional analysis of cardiac‐specific loss of MuRF1 in diabetic cardiomyopathy
Authors: Willis Monte S. , Rajalingam Sahith , Klüppel Michael
Published: April 2020

Diabetic cardiomyopathy is a serious complication in patients with type 2 diabetes, leading to pathological remodeling of the heart and a significantly increased risk of heart failure. Cardiovascular diseases are the leading cause of death in type 2 diabetes patients. The E3 ubiquitin ligase MuRF1 is expressed in heart and skeletal muscle, and we have shown that differential ubiquitination of target proteins by MuRF1 is of critical importance for the heart’s responses to atrophic and hypertrophic stimuli. In recent in vitro work, we showed that MuRF1 regulates cardiomyocyte metabolism by mono‐ubiquitination and subsequent nuclear export of PPARα, a master regulator of peroxisome function and fatty acid oxidation. These results suggest that MuRF1 plays a so far unknown role in heart metabolism and the progression of diabetic cardiomyopathy. However, any tissue‐specific roles of MuRF1 in heart versus skeletal muscle in‐vivo have not been experimentally addressed so far. To analyze the function of MuRF1 specifically in the heart during diabetic cardiomyopathy, we generated a mouse strain carrying a conditional and inducible loss‐of‐function mutation in MuRF1 via the introduction of loxP sites (MuRF1‐flox) and crossed these animals to transgenic animals expressing Cre recombinase under the control of the cardiomyocyte‐specific αMHC promoter (αMHC‐MerCreMer). Mice were treated with tamoxifen in chow for 7 days, followed by 21 days of chow. Subsequently, diabetic cardiomyopathy was induced by a 60% high fat diet (HFD) for 26 weeks, and disease progression was monitored by echocardiography, electrocardiography, glucose tolerance test and DEXA scans. Our preliminary data show that cardiac loss of MuRF1 can exacerbate cardiac deficits associated with diabetic cardiomyopathy. We will present data on our ongoing phenotypic analysis of these animals, and the elucidation of causative molecular mechanisms, including the effects on PPARα localization/activity and peroxisome function. This analysis will shed light on novel cardiac‐specific roles of MuRF1 in the context of diabetic cardiomyopathy, and potentially reveal new functions of cardiac MuRF1 in mediating crosstalk between heart and other tissues metabolically altered in diabetes, including skeletal muscle, liver, and adipose tissue.

Support or Funding Information

National Heart, Lung, and Blood Institute, R01HL104129
Mitochondrial Localization and Function of SESN2
Authors: Budanov Andrei V. , Kovaleva Irina , Tokarchuk Artem , Zheltukhin Andrey O. , Dalina Alexandra A. , Lyamzaev Konstantin G. , Haidurov Alexander , Chumakov Peter M.
Published: April 2020

The goal of this study was to understand the potential mechanisms responsible for the regulation of metabolism and mitochondrial functions by SESTRIN2 (SESN2) and other members of the SESTRIN protein family. Our studies are based on cell fractionation and microscopy analyses to determine the localization of SESN2 and on theC. elegans model to define the role of SESTRINs in mitochondrial respiration.

SESN2 is a member of evolutionarily conserved SESTRIN protein family found in the majority of theMetazoa species. SESN2 is transcriptionally activated by many stress factors including metabolic derangements, oxidants, and DNA‐damage. As a result, SESN2 suppresses oxidative stress, tunes up metabolic pathways, and regulates cell viability. Many of the activities of SESN2 are linked to regulation of mechanistic target of rapamycin complex 1 kinase (mTORC1) that plays the central role in the regulation of cell growth, metabolism, and autophagy. SESN2 inhibits mTORC1 activity through the interaction with the GATOR2 protein complex that prevents the inhibitory effect of GATOR2 on the GATOR1 protein complex and relieves an inhibitory effect of GATOR1 towards RagA/B GTPases, the major activators of mTORC1. Despite the well‐established role of SESN2 in the mTORC1 inhibition, some other SESN2 activities are not well characterized. As established by us and other groups, SESN2 can control mitochondrial respiration and cell death via mTORC1‐independent mechanisms. We hypothesized that these processes can be explained by the direct effects of SESN2 on mitochondria. To study the potential localization of SESN2 on mitochondria we performed mitochondrial fractionation from different human cancer cell lines and observed that considerable amounts of SESN2 protein were co‐purified with mitochondria. According to the results of treatment with proteases, SESN2 was located on the outer mitochondrial membrane. While we observed no association of SESN2 with the AKAP1 protein, as was previously reported, we found that the mitochondrial localization of SESN2 depends on GATOR2. We determine that mitochondrial SESN2 might be responsible for the regulation of the accumulation of reactive oxygen species and respiration in mammalian cells andC. elegans.

We have shown that SESN2 is associated with mitochondria and can be directly involved in direct regulation of mitochondria functions.

Support or Funding Information

This work was supported by Russian Science Foundation grant 17‐14‐01420.
Activation of Transcription Factor Nrf2 Ameliorates Age‐Associated Decline in Kidney Function
Authors: Mohammad Razia Sultana , Memon Rabia , Banday Anees , Lokhandwala Mustafa
Published: April 2020

Aging has a strong association with the development of kidney disease. About one in four women and one in five men, aged 65 years and above, have chronic kidney disease (CKD). Studies indicate compromised mitochondrial function in aging kidneys. However, the mechanisms of the decreased mitochondrial function in aging is unclear. The present study aims to investigate the mechanisms relating to the mitochondrial dysfunction in aging. The mechanisms thus explored will help us identify novel therapeutic targets to prevent or reverse kidney damage in elder individuals. Nuclear transcription factor Nrf2 is a cytosolic protein which when activated will translocate to the nucleus. After being translocated to the nucleus, it regulates the expression of genes involved in antioxidant and detoxification mechanisms. It binds to Antioxidant Response Element (ARE) on the gene promoters and increases the expression of genes that regulate the function of cells. We previously showed that Nrf2, was decreased in the kidneys of aged rats. Additionally, we also found a decrease in mitochondrial activity and kidney function. Therefore, we hypothesized that Nrf2 regulates kidney function via improving mitochondrial function during aging. We tested this hypothesis, by using Fischer344 rats, animal model of aging, and human kidney cells (HK2) in culture. Aged male Fischer 344 (F344) rats (20–24 months old) were divided into two groups. One group was treated with Nrf2 activator, sulforaphane (SF) [15mg/kg/day in drinking water for four weeks] and the other group (Control) was given drinking water alone. Nrf2 activation had significantly reduced the oxidative stress marker 8‐isoprostane levels in urine and increased urinary antioxidant capacity in the aged rats compared to their controls. The increased levels of protein in urine and plasma creatinine in aged animals were also reduced by sulforaphane treatment. Human kidney cells (HK2) were treated with sulforaphane (SF) at a dose of 20uM in the presence and absence of H2O2. H2O2 treatment significantly reduced Nrf2 expression which was reversed by SF treatment. Also, the mitochondrial membrane potential improved in H2O2+SF treated cells as compared to H2O2 treated cells. In conclusion, our results suggest that Nrf2 activation plays a key role in improving kidney function in aged rats.

Support or Funding Information

NIH Aging grant: AG057024‐01A1
Baroreflex Sensitivity in Facioscapulohumeral Muscular Dystrophy
Authors: Coffman Shandon , Larson Mia , Vera Kathryn , Lee Emma , McConville Mary , Keller-Ross Manda
Published: April 2020

Facioscapulohumeral muscular dystrophy (FSHD), the third most common muscular dystrophy, is caused by a genetic mutation that alters DUX4 gene expression. Although cardiac muscle is known to be spared in people with FSHD, they can develop significant morbidities and physical disabilities because of their reduced mobility. Autonomic dysregulation via heart rate variability has been demonstrated in people with FSHD. It is unknown, however, if baroreflex function, an important regulator of blood pressure, is impaired in people with FSHD.

OBJECTIVE

To examine if baroreflex sensitivity is impaired in patients with FSHD.

METHODS

Thirty minutes of resting blood pressure, heart rate, and baroreflex sensitivity (via the sequence method) was measured in 7 patients with FSHD (age: 48 ± 8 yrs, avg ± SD) and 7 sex‐ and age‐matched controls (age: 40 ± 7 yrs, p > 0.05).

RESULTS

People with FSHD were less active (FSHD Activity Metabolic Index (AMI): 17.8 ± 23.0 kcal/day; controls AMI: 151 ± 105 kcal/day; p = 0.01), but had a similar body mass index compared to controls (FSHD: 25 ± 4; controls: 26 ± 3; p > 0.05). Baroreflex sensitivity was similar among FSHD participants and controls (FSHD: 12.6 ± 8.3 ms/mmHg; controls: 16.6 ± 9.6 ms/mmHg; p = 0.40). Mean arterial pressure was also similar for FSHD participants compared to controls (FSHD: 95.3 ± 6.1 mmHg; controls: 91.6 ± 8.6 mmHg; p = 0.37). Heart rate was elevated in FSHD participants compared to controls (FSHD: 66.6 ± 7.5 BPM; controls: 56.8 ± 6.2 BPM; p = 0.03).

CONCLUSIONS

These findings suggest that baroreflex regulation is not diminished in people with FSHD, but an elevated heart rate may indicate physical deconditioning due to limited mobility.

Support or Funding Information

Scholarly Inquiry at TJU, Greg Marzolf Jr. Foundation, Friends of FSH Foundation.
Protein Fractionation Methods Applied to the Delineation of Bioactive Proteins from the Zebra Mussel, Dreissena polymorpha.
Authors: Dachel Gabriel , Grant Jennifer
Published: April 2020

Dreissena polymorpha is an invasive species that has infested the Great Lakes, portions of the Mississippi River, and roughly 250 lakes in Wisconsin. To date, there is no real ability to manage zebra mussels; efforts are limited to preventing the Zebra mussels from spreading further into Wisconsin. However, some groups in Europe have turned to the zebra mussel as a source for livestock feeds (McLaughlin, 2014.) In a tangential line of research, there is much interest in the study of marine shelled organisms, including abalone and crab, as sources of bioactive proteins. We have methods commonly used to understand the proteome of the shells of marine organisms to understand the protein content of the shells of zebra mussels. This poster reports on efforts to fractionate proteins and peptides as the first step towards understanding the antibiotic potential of zebra mussel shell extracts. Ammonium sulfate fractionation methods, as well as ion exchange chromatography and lectin chromatography, were investigated.

This work may lead to the discovery a useful protein that creates a need for a bivalve that is otherwise an invasive pest in the United States.
Potential biochemical mechanisms of the dual sigma‐1 and dopamine transporter ligand CM699
Authors: Hong W. Conrad , Sharma Abhisheak , Hiranita Takato , Avery Bonnie A. , McCurdy Christopher R
Published: April 2020

CM699 is a novel compound with dual action at the sigma‐1 receptor (σ1R) and the dopamine transporter (DAT). A previous study showed that CM699 dose‐dependently produced insurmountable antagonism of cocaine self‐administration in rats and blunted cocaine‐induced dopamine increase in rat nucleus accumbens shell (Katz et al. EB 2014 Abstract #803.3). Following a single intravenous administration, CM699 (5 mg/kg) showed extensive extravascular distribution (Vd, 8.4 ± 0.8 L/kg) and low clearance (1.3 ± 0.1 L/h/kg) with negligible extra hepatic elimination in male rats. CM699 showed adequate systemic exposure with elimination of half‐life of 4.4 ± 0.1 h. Here we explored potential biochemical and pharmacological mechanisms underlying the effects of CM699. A biochemical method on multimerization of σ1R in transfected cells was employed to differentiate σ1R agonists and antagonists. Similar to σ1R antagonists (CM304, haloperidol and BD1063), CM699 significantly increased σ1R multimers. In contrast, the σ1R agonist, (+)‐pentazocine, decreased σ1R multimers. We further examined modulation of cocaine‐induced DAT conformational changes by σ1R ligands, using cysteine accessibility assays in cell models co‐expressing DAT and σ1R. It has been shown previously that upon cocaine binding, side chain accessibilities of residues 306 and 316 in transmembrane domain 6a (TM6a) of DAT are enhanced in a dose‐dependent manner, and preincubation with σ1R agonist (+)‐pentazocine potentiates cocaine’s effects. CM699 appeared to block the potentiation by (+)‐pentazocine on cocaine‐induced cysteine accessibility changes in TM6a of DAT. Together with data from earlier studies, these results confirm that CM699 is a σ1R antagonist. Further, they provide potential mechanistic insight on CM669’s ability to abolish cocaine self‐administration in rodent models, and strengthen the hypothesis that compounds with dual σ1R/DAT actions may be explored as a novel approach to medication development for cocaine addiction.

Support or Funding Information

Butler University Faculty Startup Fund, R01 DA023205 (CRM), NIH intramural Research Program