bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationNational 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 InformationSupport: 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 InformationAlaska 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 InformationNational 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 InformationThis 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 InformationNIH 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.OBJECTIVETo examine if baroreflex sensitivity is impaired in patients with FSHD.METHODSThirty 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).RESULTSPeople 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).CONCLUSIONSThese 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 InformationScholarly 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 InformationButler University Faculty Startup Fund, R01 DA023205 (CRM), NIH intramural Research Program
The Role of Pigment Epithelium‐Derived Factor’s Anti‐Angiogenic Properties on Tumor Suppression
Authors: Wagoner Natalie , Welker Blake , Muralidharan Mahathi , Weltert Gioia 
Published: April 2020

Pigment epithelium‐derived factor (PEDF) is a multimodal secreted glycoprotein of roughly 50kDa size and 418 amino acids in length and is found in the interphotoreceptor matrix (ICM) that holds a variety of biological functions. It is a member of the non‐inhibitory serpin family and was first identified as a neurotrophic factor in retinal pigment epithelial cells, however, it has also been identified in several tissues throughout the body including the heart, lung, brain, and pancreas. PEDF is highly conserved throughout evolution and is known to possess neurotrophic, neuroprotective, anti‐tumorigenic, anti‐metastatic properties, and anti‐angiogenic properties. Indeed, PEDF is known as one of the most potent naturally occurring anti‐angiogenic agent. Angiogenesis plays a critical role in the development of cancer because it stimulates the formation of new blood vessels which carry blood to the tumor thereby providing it unlimited access to critical nutrients and oxygen needed for sustained growth and metastatic spread. Hence, the ability of PEDF to block angiogenesis highlights its potential as a novel anti‐cancer agent. Apart from inhibiting angiogenesis, PEDF also inhibits cancer cell proliferation and induces programmed cell death (apoptosis) through activation of FasR/FasL death signaling pathway. The importance of PEDF in cancer is validated by the fact that its expression is significantly reduced in several types of cancers and that loss of PEDF expression is associated with aggressive disease and poor overall survival. Indeed, PEDF expression has been shown to be markedly reduced in breast tumors compared with normal breast tissue and loss of PEDF is associated with resistance to anti‐endocrine therapy due to activation of two critical breast cancer proteins; the estrogen receptor (ER) and the Rearranged during Transfection (RET) proteins. The unique biology of the PEDF protein is directly related to its structure. PEDF is asymmetrically charged, one side being highly acidic the other highly basic, giving it a polar structure that has two functional epitopes. The acidic region contains a 34‐mer peptide that interacts with the PEDF‐RA receptor identified in endothelial cells. When this interaction occurs, apoptosis is initiated, epithelial cell migration is blocked, and corneal angiogenesis is induced. On the other hand, the basic region consists of a 44‐mer peptide that interacts with the PEDF‐RN receptor which has been identified on Y‐79 cells, cerebellar and motor neurons, and in the neural retina. When this epitope is activated, PEDF assists in conducting neural differentiation.Support or Funding InformationThe Olathe North MSOE Center for BioMolecular Modeling SMART Team used 3‐D modeling and printing technology to examine structure‐function of PEDF