bluuebunny/crossref_metadata_2025_split huggingface dataset:
Does DUSP15 promote activation of ERK MAPK signaling after Hsp90 inhibition in the spinal cord to promote opioid anti‐nociception?
Authors: Gabriel Katherin , Duron David , Streicher John , Langlais Paul , Barker Nathalie 
Published: April 2020

The misuse of opioids has become a prevalent problem for chronic pain relief, worsened by numerous side effects such as constipation, tolerance and addiction. Researchers have attempted to optimize opioids to decrease side effects and eliminate dependence by modulating the signal transduction cascades of the mu opioid receptor (MOR). Previous studies from our lab have demonstrated that the chaperone protein Heat shock protein 90 (Hsp90) is an important regulator of the ERK/MAPK pathway which is downstream of MOR. When an Hsp90 inhibitor is administered directly to the spinal cord, there is an increase in opioid antinociception in vivo, signifying that Hsp90 acts to repress the ERK/MAPK signaling pathway and reduce antinociception in the spinal cord. The exact molecular mechanism including the key players in activating opioid anti‐nociception in the spinal cord are still unknown and need to be further investigated. We thus performed quantitative proteomic analysis on the spinal cords of male and female CD‐1 mice treated with the Hsp90 inhibitor 17‐AAG. Network analysis of this data revealed that the phosphatase DUSP15 was upregulated; DUSP15 is known to be a key activator of ERK MAPK, suggesting this molecule could be the mechanism by which spinal Hsp90 inhibition activates ERK MAPK and thus opioid anti‐nociception. To test this hypothesis, we will perform CRISPR/Cas9 gene editing in the spinal cords of adult male and female CD‐1 mice to knock down DUSP15. We will then combine knockdown with Hsp90 inhibitors (17‐AAG, KU‐32) and opioids in the tail flick anti‐nociception assay, to reveal if DUSP15 knockdown will affect the enhanced anti‐nociception caused by spinal Hsp90 inhibition. These studies will thus reveal a key molecular mechanism by which Hsp90 regulates the spinal cord opioid system. Our other studies have shown that spinal Hsp90 inhibition causes enhanced anti‐nociception and decreased side effects like tolerance; these studies will also establish the basic science foundation for the use of Hsp90 inhibitors to improve opioid therapy during chronic pain.Support or Funding InformationThese studies were supported by an Arizona Biomedical Research Commission New Investigator Award (#ADHS18‐198875) and institutional funds from the University of Arizona to JMS. The authors have no other relevant conflicts of interest to declare.
zDHHC9‐mediated palmitoylation promotes hypertrophic signaling in the heart
Authors: Essandoh Kobina , Subramani Arasakumar , Molkentin Jeffrey , Brody Matthew 
Published: April 2020

Heart disease remains a leading cause of mortality in the United States, contributing to over 600,000 deaths per year in the United States. Notwithstanding, the molecular mechanisms underlying the pathophysiology of heart disease remain largely elusive. Receptors, ion channels, and signaling proteins that regulate cardiac contractility and physiology are modified by post‐translational modifications such as phosphorylation. Recent research into the roles of S‐acylation or palmitoylation, the reversible post‐translational modification of proteins with fatty acids, has demonstrated critical functions in the regulation of intracellular signaling in physiology and disease. Palmitoylation, which is catalyzed by the zinc finger Asp‐His‐His‐Cys (zDHHC) family of S‐acyl transferases, has been implicated in trafficking, stability, sorting and binding of proteins to cofactors. However, the roles of palmitoylation in cardiac signal transduction and cardiac pathogenesis are not well understood. In HEK‐293 cells, the Golgi‐localized acyl transferase, zDHHC9, was shown to palmitoylate and induce plasma membrane localization and activation of H‐Ras and N‐Ras, members of the Ras family of membrane‐localized GTPases. Activation of Ras enhances the activity of downstream pathways such as the ERK/MAP kinase pathway and c‐Jun N‐terminal kinases (JNK), which induce pathological cardiac remodeling in the heart. To investigate the potential roles of zDHHC9 in cardiac pathogenesis, we generated a transgenic mouse model with cardiomyocyte‐specific overexpression of zDHHC9 (Tg‐Zdhhc9). These mice exhibited cardiac hypertrophy with increased ventricular wall thickness at 6 months of age. At 8 months of age, Tg‐Zdhhc9 mice showed impaired cardiac contractile function and severe dilated cardiomyopathy. Interestingly, there were no significant differences in palmitoylation of H‐Ras in Tg‐Zdhhc9 hearts compared to control hearts, indicating that zDHHC9‐triggered cardiac hypertrophy may be due to palmitoylation of other substrates in cardiomyocytes. Ongoing work is utilizing unbiased quantitative palmitoyl proteomics to identify novel zDHHC9 substrates that mediate activation of hypertrophic signaling in the cardiomyocytes. Altogether, these data suggest zDHHC9‐mediated palmitoylation contributes to hypertrophic signaling and heart disease.Support or Funding InformationThis work was supported by National Heart, Lung and Blood Institute grant R00‐HL‐36695 (MJB).
The Gantzer Muscle: An Expanded Study of this Variant Forearm Muscle
Authors: Herrold Christina B. , Cook Ryan L. , Burkett Jesse T. , Hobeika Natalie A. , Zdilla Matthew J. , Lambert H. Wayne 
Published: April 2020

Gantzer muscles are anatomical variations found within the flexor compartment of the forearm. The Gantzer muscle typically arises from the flexor digitorum superficialis (FDS) and inserts into the flexor pollicis longus (FPL) or flexor digitorum profundus (FDP). The presence of this muscle can cause various compressive injuries, including neuropathy of the median or anterior interosseous nerve and impingement of the ulnar, common, or anterior interosseous arteries. Despite its high prevalence, the Gantzer muscle is often excluded from the differential for acute compartment syndrome and should be further considered during treatment and surgical management. This study expanded upon a previous assessment of cadaveric specimen forearms to determine further the prevalence of origin, insertion, and innervation of Gantzer muscles, as well as possible compressions induced from impingement by the anomalous muscle. A total of 288 limbs were dissected in 144 cadavers. The Gantzer muscle was found in 54.6% (148:271) of limbs. Therefore, in general, the Gantzer muscle is more likely to be present than absent. This report also details common and uncommon origins and insertions of Gantzer variants.Support or Funding InformationT. Walley Williams Summer Anatomy Research Fellowship; WV Research Challenge Fund [HEPC.dsr.17.06]
A new approach for analyzing pHi recoveries from NH4+‐induced acid loads, applied to rat hippocampal neurons and astrocytes
Authors: Ruffin Vernon , Boron Walter 
Published: April 2020

Regulation of intracellular pH (pHi) in brain cells (neurons and neuroglia) is essential for optimal cell function and primarily maintained by acid/base transporters in the SLC9 and SLC4 families. Large deviations in pHi can lead to disrupted cellular function or even cell death, whereas small changes have been proposed as a signaling method due to the pH‐sensitive residues within protein structure. In the hippocampus (HC), tight regulation of pHi underlies the cascading reactions and impacts the structure of proteins necessary for learning and memory formation. The present work provides a detailed examination of the methods used in the analysis of steady‐state pHi and the recovery from NH4/NH3 induced acidosis by the SLC9s in the absence of CO2/HCO3−, when SLC4 (HCO3−‐sensitive) acid/base transporters are largely inactive. In the present study, we use the pH‐sensitive dye BCECF to examine HC neurons and astrocytes, co‐cultured from embryonic (E18–20) Sprague Dawley rats. In the protocol, two sequential NH4/NH3 pulses are delivered and the steady‐state pHi values is identified before (checkpoint C) and after (E for pulse 1 and F for pulse 2) the NH4/NH3 pulse. Typically, steady‐state pHi values at C were more alkaline than E and F, most likely due to the inactivity of HCO3−‐dependent acid loaders in the SLC4 family. The recovery from acidosis is fit with a double exponential (DExp) which we replot as dpHi/dt vs pHi. With this traditional approach, dpHi/dt, as it approaches the asymptotic pHi, becomes slightly non‐linear. To exploit the mainly linear portion of the dpHi/dt vs. pHi plot (from the DExp fit), we fit these dpHi/dt vs. pHi points with a single exponential (SExp) to produce a quasi–single‐exponential rate constant. This analysis—when transformed to the pHi vs. time domain—generally produces a very good fit to the original pHi vs. time data. We summarize the twin pHi recoveries from individual experiments in thumbnails in which we display the quasi–single‐exponential dpHi/dt line segments that represent the pHi recoveries from the first and second NH3/NH4+ pulses. For each line segment in the thumbnail, the slope represents the quasi–single‐exponential rate constant, and the projected pHi‐axis asymptote represents the predicted new steady‐state pHi. The thumbnails permit a quick assessment of the pHi recoveries from acidosis in many cells, including a sense of the variability between the first and second pulses, or the variability among cells. The advantage to this approach is that our method of analysis allows an entire population of cells to be examined individually but quickly.Support or Funding InformationThis work was supported by the NIH National Heart, Lung, and Blood Institute. Grant # R01 NS018400
Studies on the Interaction of Unfractionated Heparin and Sulodexide with Functional Antiheparin Platelet Factor 4 Antibodies as Studied in Platelet Aggregation Assays
Authors: Farooqui Ambar , Hoppensteadt Debra , Iqbal Omer , Bacher Cassandra , Lewis Mary , Walenga Jeanine , Kouta Ahmed , Jeske Walter , Fareed Jawed 
Published: April 2020

IntroductionSulodexide represents a glycosaminoglycan derived antithrombotic agent which is clinically used for the management of vascular disorders and is pharmacologically active after both parenteral and oral administration. The purpose of this study is to compare the relative effect of sulodexide in mediating heparin induced thrombocytopenia (HIT) antibodies induced aggregation of human platelets in a standardized test system which is developed to compare the effects of heparins and related drugs.Materials and MethodsActive pharmaceutical ingredients (API versions of sulodexide), fast moving heparin and dermatan sulfate were obtained from Alfa Sigma (Bologna, Italy). API versions of UFH were obtained from Medefil, Inc (Glendale Heights, IL, USA). All agents were dissolved in saline at a stock concentration of 1 mg/ml. Working dilutions were prepared at 100, 10 and 1 mg/ml. The source of functional HIT antibody represented a frozen pool of aphresis fluid (AF) collected from five clinically confirmed heparin induced thrombocytopenia patients. The functionality of the HIT antibodies in this pool were confirmed in the platelet aggregation and 14C serotonin release assay. All results were compiled in terms of percent aggregation of platelets and represented as mean ± standard deviation.ResultsUFH produced a concentration dependent aggregation from 0 – 10 ug/ml reaching a peak aggregation of 60 ± 18.76%. Sulodexide produced a relatively weaker aggregatory response and produced mild aggregation in the same concentration range exhibiting comparable platelet aggregation at 10 ug/ml. The fast moving heparin component of sulodexide produced a concentration dependent response comparable to heparin reaching a maximum aggregation of 66 ± 30.67% at 10 ug/ml. The dermatan sulfate component did not produce any significant aggregation of platelets and no concentration dependence was observed (<10% aggregation). Wide intra variations were observed in the HIT antibody aggregation response with both the heparin and sulodexide but not with dermatan sulfate.DiscussionThese results clearly show that in comparison to heparin, sulodexide produces a relatively weaker response in the functional HIT antibody screening studies. However, the fast moving heparin component of sulodexide produces comparable responses to UFH whereas dermatan sulfate has much weaker aggregation response. The presence of dermatan sulfate may modulate the interaction between platelet factor 4 and sulfated polysaccharide change present in sulodexide preparations.
Influence of the eye on neurocranium development; Insights from Mexican cavefish (Astyanax mexicanus)
Authors: Gimhani Nuwanthika , Atukorallaya Devi 
Published: April 2020

Astyanax mexicanus is being recognized as an intriguing teleost model in developmental biological studies. A. mexicanus exists as two morphs eyed, pigmented surface dwelling morph known as surface fish and an unpigmented blind cave dwelling cavefish. The midline bones of anterior neurocranium of A. mexicanus consists of parasphenoid, ethmoid and vomer bone. The detailed anatomical development of neuorocranial skeleton of this fish is unexplored thus far. The present comparative study is designed to understand the palatal bones, parasphenoid, ethmoid and vomer bone development among surface fish and in two population of cavefish Pachon and Tinaja. Cavefish and surface fish (Astyanax mexicanus) were included in to this study. Fish were fixed at different time point ranging from 10 dpf, 20 dpf, 35dpf, 60 dpf and 5 year old adults. Alizarin red bone stained were used to stain the skeleton. Dissected samples were examined under the stereo microscope for the ossification sequence of the palatal bones. The micro‐CT images of adult fish were taken using SkyScan micro‐CT imager (Bruker) and CT vox software (Bruker) was used for image analysis. First ossification center appears at the parasphenoid bone at 5 dpf. The anterior neurocranial bones ossified via endochondral ossification. Both cavefish and surface fish ossification starts from the ossification center locate in the center of the parasphenoid cartilaginous palate. Ossification progresses towards anterior direction and merge with the ossifying maxillary bone. The ossification extend up to the notochord in posterior direction. The Micro‐CT images showed that the morphology of parasphenoid bone is different in each eye morph. In surface fish parasphenoid bone curved and it has 20° angle of elevation towards the anterior direction. In contrast, parasphenoid bone found to be straight in Pachon and Tinaja cavefish. Absence of eye structures have an influence on determining the shape of parasphenoid bone.Support or Funding InformationWe would like to thank the Natural Sciences and Engineering Research Council of Canada (#2019‐05364) for funding this research. We acknowledge the support from University of Manitoba.
Interplay Between Whole Body Metabolism, Physical Activity, and Muscle Function Following Volumetric Muscle Loss Injury
Authors: Dalske Kyle A. , Basten Alec M. , Raymond-Pope Christiana J. , Call Jarrod A. , Greising Sarah M 
Published: April 2020

Volumetric muscle loss (VML) injury occurs when a substantial volume of skeletal muscle is abruptly removed and results in significant long‐term functional limitations. Following VML injury, patients likely undergo periods of low or no mobility and are expected to have reduced physical activity. Immobility and lack of physical activity leads to impaired muscle function, and increased all‐cause mortality and risk for chronic disease. It is plausible that inactivity and immobility negatively affect outcomes following VML injury, exacerbating functional impairments. Our previous work supports an ~25% lower oxygen consumption rate in permeabilized muscle fibers of the muscle remaining after VML up to 4 months post‐injury, but how this may impact whole body metabolism is unclear. Therefore, the purpose of this study was to characterize whole body metabolic and physical activity following VML injury. We hypothesized that metabolism and physical activity would be impaired following VML. To elucidate the interplay between metabolism, activity, and muscle function following VML injury, adult male C57BL/6 mice (n=7) underwent a full‐thickness multi‐muscle VML injury (removal of ~22mg) to the gastrocnemius, soleus, and plantaris, or sham surgery (n=7). Twenty‐four hour whole body metabolic and physical activity was assessed pre‐, and 2 and 6 weeks post‐VML (or sham) injury. Contractile activity of the ankle plantarflexors was assessed terminally at 8 weeks post‐VML. Prior to randomization into experimental groups metabolic rate was ~18.5kcal/kg/hr and there was no difference in any metabolic or activity measure (p≥0.21), and both groups gained ~3g over the 8 week period. Whole body metabolism was evaluated for the 24‐hour period and isolated by the 12 hours of active and inactive time; respiratory exchange ratio (RER), an indirect measure of muscle oxidative capacity, was not different across experimental groups (p≥0.76) but was significantly decreased with time (main effect p≤0.01). Two weeks post‐VML mice tended to become hypermetabolic with a metabolic rate ~14% greater than sham. On average, mice ambulated ~1.2 km per 24‐hour period and contrary to the hypothesis, there was no difference in activity levels (p≥0.23) between sham and VML injured mice. Terminally the plantarflexor muscle group of the VML‐injured mice had ~1.4 fold greater maximal passive torque (indicator of muscle stiffness) and ~40% less maximal active isometric torque. These findings indicate limited changes whole body metabolic and physical activity levels in a model of VML injury, contrary to our hypothesis. Future studies will limit physical activity levels in this model to better recapitulate the low or no mobility expected following VML injury clinically.Support or Funding InformationSupported by W81XWH‐18‐1‐0710 & T32AR050938
CD99L2 is a Major Regulator of Transendothelial Migration
Authors: Rutledge Nakisha S. , Sullivan David , Muller William 
Published: April 2020

Transendothelial Migration (TEM), the process in which leukocytes leave the bloodstream to enter inflamed tissues, is a crucial step in the inflammatory response. Adhesion/signaling molecules such as PECAM and CD99 are important for the initiation and completion of TEM, respectively. We and others have shown that CD99‐like 2 (L2), a highly glycosylated 52 kD type‐1 membrane protein that is the only molecule in the genome related to CD99, is also important for TEM. However, L2 is different enough from CD99 that it is not clear whether it regulates TEM by the same mechanism as CD99. Using intravital microscopy, we show a critical role for L2 in recruitment of monocytes and neutrophils to sites of inflammation. An acute dermatitis model shows L2 specifically facilitates leukocyte extravasation across endothelial cell borders.All of the published studies on L2 have been performed in mice. In order to study the mechanism used by L2 and the relevance to human inflammation, we studied the role of L2 on human cells in vitro. Our data show that similar to PECAM and CD99, human L2 is constitutively expressed at the borders of endothelial cells and on the surface of leukocytes. Blocking L2 using antibodies or knockdown of L2 significantly reduced transmigration of human neutrophils and monocytes across human endothelial cells. TEM is restored by re‐expression of L2. Blockade of either leukocytes or endothelial cells blocks equivalently to blocking both. TEM is significantly reduced in the presence of soluble L2‐Fc. These data are consistent with a homophilic interaction between leukocyte L2 and endothelial cell L2 being essential for TEM. Our data also show that L2 regulates a step in TEM after PECAM and before CD99. Consistent with this, preliminary data suggest that L2 can promote TEM by engaging either the signaling pathway used by PECAM or the one used by CD99. Similar to PECAM and CD99, L2 promotes transmigration by recruiting the lateral border recycling compartment to the site of TEM, downstream of PECAM‐mediated initiation of TEM. Ongoing studies are focused on identifying signaling pathways used by L2 to promote TEM and validating them in vivo using intravital confocal microscopy to study TEM in real time.Support or Funding InformationT32‐5T32AI007476‐19,R01‐HL064774, and F31‐HL131355‐01
Noncompetitive allosteric antagonism of death receptor 5 by an affibody discovered via yeast display
Authors: Vunnam Nagamani , Szymonski Sophia , Petra Hirsova , Gores Gregory James , Hackel Benjamin , Sachs Jonathan 
Published: April 2020

Fatty acid‐induced upregulation of death receptor 5 (DR5) and its cognate ligand, tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL), promotes hepatocyte lipoapoptosis, which is a key mechanism in the progression of fatty liver disease. Accordingly, inhibition of DR5 signaling represents an attractive strategy for treating fatty liver disease. Ligand competition is prevalent in tumor necrosis factor receptor antagonism, but recent studies suggest a compelling alternative: non‐competitive inhibition through perturbation of receptor conformation. We used a yeast‐displayed combinatorial library to identify a synthetic affibody that specifically binds DR5. Biophysical and biochemical studies show that the affibody neither blocks TRAIL binding nor prevents the receptor‐receptor interaction. Live‐cell fluorescence lifetime measurements indicate that the affibody induces a conformational change in transmembrane dimers of DR5 and favors an inactive state of the receptor. The affibody inhibits apoptosis in TRAIL‐ or palmitate‐treated Huh‐7 cells, an in vitro model of fatty liver disease. Thus, this lead affibody serves as a potential drug candidate, with a unique mechanism of action, for fatty liver disease.Support or Funding InformationNational Institutes of Health Grants R01EB023339, R01DK41876, R35GM131814 and R01EB028274.