bluuebunny/crossref_metadata_2025 huggingface dataset:
Transient receptor potential vanilloid 4 (TRPV4) channels are essential for alveolar epithelial cell function
Authors: Dietrich Alexander , Chao Yu-Kai , Kannler Martina , Krasteva-Christ Gabriela , Rajan Suhasini , Yildirim Ali Oender , Brosien Monika , Schredelseker Johann , Weissmann Norbert , Grimm Christian , Gudermann Thomas W. , Weber Jonas
Published: April 2020

Ischemia‐reperfusion‐induced edema formation as one of the most common and significant causes of morbidity and mortality after lung transplantation can be mimicked ex‐vivo in isolated perfused mouse lungs (IPL). Transient receptor potential vanilloid 4 (TRPV4) is a non‐selective cation channel expressed in lung endothelium where increased channel activity has been shown to compromise endothelial barrier function. Here we show enhanced edema formation in TRPV4‐deficient (TRPV4−/−) IPL compared to wild‐type (WT) controls in response to ischemia and reperfusion, indicating a protective role of TRPV4 to maintain the alveolar epithelial barrier. By immunohistochemistry or mRNA profiling, we detected TRPV4 in bronchial epithelium, alveolar type I (ATI) and alveolar type II (ATII) cells. Genetic ablation of TRPV4 resulted in reduced expression and plasma membrane insertion of the water conducting aquaporin‐5 (AQP‐5) channel in ATI cells compared to WT mice. Analysis of isolated primary TRPV4‐deficient ATII cells revealed a reduced expression of surfactant protein C (SP‐C), which is essential for decreasing surface tension and alveolar fluid homeostasis. The TRPV4 activator GSK1016790A induced increases in current densities only in WT but not in TRPV4−/− ATII cells. Although the ability of TRPV4−/− ATII cells to differentiate to ATI cells was unchanged, epithelial mesenchymal transition (EMT) of TRPV4−/− ATII cells was impaired and migration and cell barrier function of TRPV4−/− ATI cells was reduced. Moreover, TRPV4−/− lungs of adult mice developed significantly larger mean chord lengths and altered lung function compared to WT lungs. Therefore, our data highlight novel essential functions of TRPV4 channels in alveolar epithelial cells and in the protection from edema formation.

Support or Funding Information

This study was funded by the Deutsche Forschungsgemeinschaft (TRR 152 (AD, GKC, CG), GRK 2338 (AD, CG)) and the German Center for Lung Research (DZL) (AD, MB, NW, TG).

Phenotypes in alveolar epithelial cells after ablation of TRPV4. See text for details.

Figure 1
Learning Anatomy Through Yoga
Authors: Sullivan Alyssa , Vasudevan Anita , Fox Glenn M. , Prall Cristina R. , Alsup B. Kathleen
Published: April 2020

The aim of this project is to provide a unique tool for anatomy students of all levels to apply common physical activities in the process of learning anatomical structures and functions. The practice of yoga was chosen to demonstrate the dynamic anatomical components of the upper and lower limbs through a variety of poses. This is explored through a series of videos: ‘Learning Anatomy through Yoga’. This series was developed with a fourth year medical student at the University of Michigan, who is also a certified yoga instructor. During the musculoskeletal sequence in April, first year medical students will assess the efficacy of this resource via surveys. In the future, the ‘Learning Anatomy through Yoga’ video series will be part of a broader project that demonstrates functional anatomy in everyday physical activity, such as weight lifting, rowing, pilates, etc.

Instructor demonstrating specific attachment points on skeleton

Figure 1
Limb blood flow and muscle oxygenation responses to rhythmic exercise below and above critical force
Authors: Hammer Shane M. , Alexander Andrew M. , Didier Kaylin D. , Huckaby Lillie M. , Barstow Thomas J.
Published: April 2020

This study aimed to determine brachial artery blood flow (Q̇BA) and microvascular oxygen delivery responses to rhythmic exercise above and below critical force (CF; the isometric analog of critical power). We hypothesized that Q̇BA, deoxygenated‐heme (deoxy‐[heme]; an estimate of microvascular fractional oxygen extraction), and total‐heme concentrations (total‐[heme]; an estimate of changes in microvascular hematocrit) would demonstrate physiological maximums above CF despite increases in exercise intensity. 13 healthy subjects performed 1) a 5‐min rhythmic isometric‐handgrip maximal‐effort test (MET) to determine CF and 2) constant target‐force tests above (severe‐intensity; S1 and S2) and below (heavy‐intensity; H1 and H2) CF. CF was 189.3 ± 16.7 N (29.7 ± 1.6 %MVC). At end‐exercise, Q̇BA was greater during S1 (418 ± 147 mL/min) and S2 (403 ± 137 mL/min) than during H1 (287 ± 97 mL/min) and H2 (340 ± 116 mL/min; p < 0.05) but was not different between S1 and S2. Further, Q̇BA for S1 and S2 were not significantly different from the Q̇BA estimated for CF (392 ± 37 mL/min). At end‐exercise, deoxy‐[heme] was not different between S1 (150 ± 50 μM) and S2 (155 ± 57 μM), but was greater during S1 and S2 than during H1 (101 ± 24 μM) and H2 (111 ± 21 μM; p < 0.05). Total‐[heme] was not different between S1 (404 ± 58 μM) and S2 (397 ± 73 μM), but was greater during S1 and S2 than H1 (352 ± 58 μM; p < 0.01) but not H2 (371 ± 57 μM). These data suggest limb blood flow limitations and maximal levels of muscle microvascular oxygen delivery and extraction during exercise above, but not below, CF.

End‐exercise brachial artery blood flow (Q̇BA) values. Q̇BA at end‐exercise below (● H1 and ○ H2) and above (▼ S1 and Δ S2) critical force (CF), and Q̇BA predicted at CF by linear regression (♦). * Significantly greater than Q̇BA at end‐exercise during H1 (p < 0.05). ** Significantly greater than Q̇BA at end‐exercise during H2 (p < 0.05). Note no differences among end‐exercise Q̇BA values at (estimated) and above CF, suggesting a maximal response in the severe‐intensity domain.

Figure 1

Frequency‐domain near‐infrared spectroscopy (FD‐NIRS) measurements of muscle oxygenation during exercise below (● H1 and ○ H2) and above (▼ S1 and Δ S2) critical force (CF). Dashed lines indicate average endexercise values of respective FD‐NIRS signals during the maximal‐effort test (MET). * Significantly different from end‐exercise during H1 and H2 (p < 0.05). ** Significantly greater than total‐[heme] at end‐exercise during H2 (p < 0.05).

Figure 2
Characterizing the Functional Role of Tyrosine 381 in the Activity of E. coli Aminopeptidase N (PepN) Through Mutagenesis
Authors: Crawford Patrick , Rivard Ryan , Nelson Nicole , Butnik Kimberley
Published: April 2020

Aminopeptidase N (PepN) from E. coli is an M1 class peptidase with broad specificity. PepN was over‐expressed, purified, and mechanistically characterized with L‐alanine‐p‐nitroanilide in an effort to understand the role of tyrosine 381. In an effort to characterize the functional roles of binding region components, this tyrosine was mutated to alanine, Y381A‐ PepN, and phenylalanine, Y381F‐ PepN. These mutants included a silent mutation, that is an addition or subtraction of an restriction enzyme site to easily identify successful mutagenesis. Successful mutants were over‐expressed, purified, and mechanistically characterized. Y381A‐PepN exhibited greater than 900‐fold loss of activity (kcat), while Y381F‐PepN maintained comparable activity, when compared to the wild‐type PepN. This suggests that Tyr381 has an essential mechanistic role in the function of PepN beyond the suggested role of hydrogen bond stabilization of substrate and product.

Support or Funding Information

This work was funded by Augustana College New Faculty Research Fund, Augustana College Research Foundation, and Augustana College Edwin Erickson Summer Research Award.

Agarose gel showing silent mutation, addition of a NarI restriction site to Y381A‐PepN.

Lanes from left to right: wild‐type, Y381A1‐PepN, Y381A2‐PepN. All samples were digested with NarI for 1 hour at 37°C. Y381A1 shows an unsuccessful mutation (lack of alteration to restriction digestion map). Y381A2 shows an successful mutation (an alteration to restriction digestion map). New restriction digest bands due to the silent mutation are below main band.

Figure 1
Oxidized Hemoglobin Causes Human Lung Microvascular Endothelial Barrier Dysfunction
Authors: Meegan Jamie E. , Tomasek Toria , Ware Lorraine B. , Bastarache Julie A.
Published: April 2020

Cell‐free hemoglobin is released into the circulation during conditions in which red blood cell lysis occurs such as hemolytic disorders and sepsis. In septic patients, increased levels of circulating cell‐free hemoglobin are independently associated with mortality and contribute to organ dysfunction, including acute respiratory distress syndrome (ARDS). Furthermore, during inflammatory conditions in which reactive oxygen species are high, cell‐free hemoglobin can be converted to higher oxidation states which enhances its damaging capacity. We previously showed that hemoglobin contributes to tissue edema in an ex vivo perfused human lung and microvascular endothelial barrier dysfunction in vitro; hemoprotein reductants (ascorbate and acetaminophen) protected against this phenomenon. Therefore, we hypothesized that higher oxidation states of hemoglobin are more injurious to the barrier function of lung microvascular endothelium. Utilizing primary human lung microvascular endothelial cells (HLMVEC), we found that stimulation with 0.5 mg/mL ferric (Hb3+; −380.2 Ω, p<0.001 vs. control) but not ferrous (Hb2+; +278.3 Ω, p=0.9893 vs. control) hemoglobin resulted in a drop in transendothelial electrical resistance (Figure
). The barrier‐disrupting effects of Hb3+ were reduced with haptoglobin (binds free hemoglobin; +208.5 Ω, p<0.0001 vs. Hb3+) and hemopexin (binds free heme), but not the iron chelator deferoxamine. The mechanism of Hb3+‐mediated microvascular endothelial barrier dysfunction involved disruption of adherens junction protein VE‐cadherin at cell‐cell borders and rearrangement of the actin cytoskeleton with intercellular gap formation. These data suggest that oxidized cell‐free hemoglobin causes microvascular endothelial barrier dysfunction to a greater degree than its non‐oxidized form.

Support or Funding Information

This work is funded by NIH HL135849, HL103836, and HL094296.

Figure 1
Anatomical Variation of High Bilateral Brachial Artery Bifurcation: A Case Report
Authors: Gupta Rohun , Pham Luu , Hsia Charlene , Daboul Rania , Galen Benjamin , Gemechu Jickssa
Published: April 2020

The brachial artery is a continuation of the axillary artery below the inferior border of the teres major muscle. It commonly bifurcates into the radial and ulnar artery low to the neck of the radius. Previous studies have documented variability for upper limb vasculature (9%–18.5%) and variability of brachial bifurcation in the middle of the arm (3–15%). However, there are a lack of cadaver studies that have discussed the effects of a brachial bifurcation in the proximal third of the arm, close to the teres major muscle. During routine anatomical dissections of 24 donors (11 males and 13 females) at Oakland University William Beaumont School of Medicine, it was found that only one donor (female) had a bilateral brachial artery bifurcation in the proximal third of the arm (4.17%). Both right and left arms were measured to be 21cm long, along with brachial artery bifurcation occurring 3cm from the teres major muscle. The bilateral bifurcation may be explained in terms of embryogenesis where there may be a persistence, unusual path, or obliteration of primitive vessels. The variance in the brachial artery bifurcation, especially in the proximal third of the arm, presents heavy clinical considerations. There is a potential that such bifurcations may affect the course of the median, radial, and ulnar nerves. Such variances may complicate surgeries in the brachial region, percutaneous brachial catheterization, and increase the likelihood of inaccurate readings of angiograms and other relevant radiological techniques. Brachial artery proximal bifurcation may also cause individuals to be vulnerable to laceration, which may result in hematomas. A proximal bifurcation of the brachial artery may affect the anastomosis of the ulnar artery with the superior and inferior ulnar collateral arteries and contribute to the excessive hematoma that was observed on the variant donor. The awareness of this clinically significant variation implicates the need for pre‐operative vessel mapping in order to decrease the possibility of iatrogenic injuries

Bifurcation of brachial artery on the left arm.

Figure 1

Bifurcation of brachial artery on the right hand. The blood was determined to be pre‐mortem, leading to a conclusion of a hematoma.

Figure 2
Diminished Expression of Na+/HCO3−cotransporter 1.2a (nbce1.2a) mRNA Accompanies Seawater Acclimation in Atlantic Salmon
Authors: Koltenyuk Victor , Nelson Nastasia , McKay Ian , Lema Sean , McCormick Stephen , Breves Jason
Published: April 2020

The life history of anadromous Atlantic salmon (Salmo salar) entails an initial freshwater stage that precedes their migration to marine environments. The development of physiological systems that will ultimately support the survival of juvenile salmon upon their entry into marine habitats is termed parr‐smolt transformation. This springtime transformation (smoltification) includes the acquisition of seawater (SW) tolerance. Thus, the ability of smolts to successfully acclimate to SW is attained through the development of branchial pathways for ion‐secretion. In addition, the capacity to rapidly attenuate ion‐absorptive processes upon exposure to SW is a vital aspect of SW tolerance. In the current study, we investigated the branchial gene expression patterns of three Na+/HCO3−cotransporter 1 isoforms (nbce1.1, nbce1.2a, and nbce1.2b) during smoltification and following seawater challenges. Given the proposed roles of Nbce1s in the absorption of environmental Na+ by branchial ionocytes in other teleosts, we hypothesized that nbce1s would be downregulated in salmon undergoing smoltification and/or following SW exposure. In two separate years, we observed springtime increases in branchial Na+/K+‐ATPase activity, Na+/K+/2Cl−‐cotransporter 1 and cystic fibrosis transmembrane regulator 1 expression characteristic of smoltification, whereas there were no seasonal changes in the expression of nbce1 isoforms. On the other hand, we observed diminished nbce1.2a expression within 2 days after the transfer of parr and smolts to SW that remained low throughout the experiment (>2 weeks). Collectively, our results suggest that while there are no clear seasonal nbce1 dynamics during smoltification, the modulation of nbce1.2a expression upon exposure to SW may underlie the attenuation of environmental Na+ uptake.

Support or Funding Information

Supported by the National Science Foundation (IOS‐1755131).
Qualitative Assessment of Carbon Nanotube Toxicity to Mouse Liver using Dark‐field Microscopy
Authors: Angtuaco Steven E. , Savenka Alena , Fite Todd , Liu Shijie , Khodakovskaya Mariya , Basnakian Alexei
Published: April 2020

Broad use of nanomaterials may eventually lead to environmental poisoning, including the contamination of food. However, methods for the assessment of nanomaterial toxicity are not well developed. The goal of the current study was to develop a techniques to measure toxicity of multiwall carbon nanotubes (MWCNTs) in vivo. For this, male CD‐1 mice were fed by oral gavage with a water suspension of MWCNTs at varying doses up to 10 mg/kg/day. Twenty‐four hours later, blood chemistry testing showed elevation of aspartate aminotransferase (AST), a liver injury marker, at the highest used dose. The liver damage was confirmed by the terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) assay. Our further analysis included the application of dark‐field microscopy (DFM) for (a) the colocalization between MWCNTs and TUNEL‐positive cells and (b) development of a method to quantify MWCNTs burden in liver tissue. DFM clearly showed some elevation of particulate material in the liver with occasional association with TUNEL‐positive cells. DFM did not seem to allow precise quantification of nanoparticle aggregates due to low sensitivity of DFM at the concentration observed in the liver. However, we discovered that CNT gavage increased the percentage of nuclei colocalized with DF objects by systematically counting the number of liver cell nuclei in contact with dark‐field (DF) objects. TUNEL‐positive nuclei were photographed, analyzed at 100‐fold magnification and sorted by CNT treatment category. The process was repeated for DAPI nuclei to serve as a control. The percentage of nuclei with DF colocalization was recorded per group. Our results indicate that the rate of colocalization between dark‐field objects and nuclei appears to correlate directly with CNT‐gavage treatment. Another important observation, confirmed by an electron microscopy, was that there were many alive TUNEL‐negative cells containing MWCNTs. Overall, our data indicate that the DFM method designed for particle toxicology has only limited potential for measurement of nanoparticle toxicity, and that some mouse liver cells are capable of accumulating MWCNTs without inducing immediate cell death.

Support or Funding Information

UAMS/ASPET Pharmacology/Toxicology Summer SURF Program USDA subcontract (Basnakian, PI) VA Merit Review grant (Basnakian, PI) COBRE grant (Hauer‐Jensen, PI)

To study the effect of carbon nanotube (CNT) ingestion on mouse liver, we systematically counted the number of liver cell nuclei in contact with dark‐field (DF) objects. We found that CNT gavage increased the percentage of nuclei colocalized with DF objects at 100x. Of a total of 38 TUNEL cells recorded in gavage tissue, 22 were touching a DF‐positive object. Livers were extracted from male CD‐1 mice 72 hours after receiving a CNT gavage of 10mg CNT/kg mouse weight.

Figure 1
Effects of Bambara Nut (Vigna subterranean) on Basal and Stimulated Gastric Acid Secretion due to Histamine, Pentagastrin and Carbachol in rats
Authors: Onwuchekwa Chinedu , Bassey Okon , Ajao Oluwatobi Sarah
Published: April 2020

The seed extracts of V. subterranean (Bambara nuts) have many therapeutic applications in folkloric medicine owing to their effective secondary metabolites such as alkaloids, flavonoids, saponins, resins, and glycosides (Andzouana et al., 2014). The infusions and decoctions of the seeds are used to treat venereal diseases, diarrhoea, cataract, beriberi and polymenorrhea (Brink and Belay, 2006; Basu et al., 2007). The raw seeds are chewed to alleviate nausea and vomiting in pregnant women. V. Subterranea seed extracts have been shown to possess some biological properties such as antioxidant and hepatoprotective (Nyau et al., 2015; Iweala and Elekwa, 2001). In spite of multi‐functional properties of V. subterranea, its gastroprotective effect has not been fully explored. This study was carried out to examine the effects of V. Subterranea on basal and stimulated gastric acid secretion (GAS) in the Wistar rats. Each group of rats consisting of 5 rats (180–210g) was treated with aqueous extract of V. Subterranea (150 mg/kg, 250mg/kg and 400mg/kg) orally for 21 days. The animals were handled in accordance with guide for the care and use of laboratory animals (NIH, 2011). At the last day of the treatment, the rats were fasted for 24 hours, anaesthetized with ketamine. GAS (mEq/l) induced by histamine, carbachol and pentagastrin administration were assessed by the continuous perfusion technique using phenolphthalein as indicator. Data obtained were expressed as the Mean ± SEM) and analyzed using one way Analysis of Variance (ANOVA) and Graph Pad prism 5. The differences in mean were considered significant at p≤ 0.05. The aqueous extract of V. Subterranea significantly decreased (p<0.05) GAS induced by the histamine, carbachol and pentagastrin in a dose‐dependent manner over 80 minutes period in all the treated groups, although at varying rates. The gastroprotective action of V. Subterranea may be multifactorial that may include an anti‐secretory action.

Support or Funding Information

We wish to thank Usmanu Danfodiyo University, Sokoto, Nigeria for their support.

Effect of Histamine on Bambara

Figure 1

Effect of Carbachol on Bambara

Figure 2

Effect of Pentagastrin on Bambara

Figure 3
TRPM5 Activity is Potentiated with Glimepiride and Acts in Tandem with KATP Channels to Stimulate Glucose‐Induced Insulin Secretion
Authors: Philippaert Koenraad , Hubert Matt , Kerselaers Sara , Light Peter E. , Vennekens Rudi
Published: April 2020

TRPM5 is a calcium‐activated monovalent cation channel expressed in the pancreatic beta‐cells where it is involved in the regulation of glucose‐induced insulin secretion. Screening of a large library of bioactive drug‐like compounds revealed that glimepiride displays TRPM5‐potentiating activity. Glimepiride is a third generation sulfolylurea drug and is used in clinical practice to stimulate insulin secretion in type II diabetic patients. It is an optimized sulfonylurea drug compound with improved pharmacological effects compared to earlier drugs in this class. One of the main advantages of glimepiride is the reduced observation of hypoglycemic events during treatment. The interaction of glimepiride with KATP channels, the bona fide target of sulfonylureas, cannot fully explain the differences in observed effects between glimepiride and earlier sulfonylureas as glyburide and tolbutamide. We used a series of in vitro and in vivo experiments to examine the interaction between glimepiride and TRPM5. We measured the TRPM5 currents evoked by glimepiride in a HEK‐cell overexpression system. In isolated pancreatic islets from WT, Trpm5−/− and KATP functional knockout mice, we measured the changes in intracellular calcium dynamics. Ultimately we observed the effects of glimepiride in vivo. Glimepiride increases TRPM5‐mediated currents in HEK cells. In isolated islets, we observe calcium activity in the presence of glimepiride in a lower concentration range compared to Trpm5−/− islets. Furthermore in islets isolated form KATP pore mutant mice, we observed increased calcium activity in the beta‐cells during application of glimepiride. Ultimately, in Trpm5−/− mice, glimepiride has less antihyperglycemic effects after a glucose injection compared to WT mice, indicating there is an important role for TRPM5 in the signal transduction of glimepiride in the beta‐cells. The action on TRPM5 is downstream of increases in [Ca2+]i, and therefore glucose‐dependent. Our data suggest an explanation for the reduced hypoglycemic effect of glimepiride. Taken together the promiscuity of glimepride leads to a synergetic action on KATP channels and TRPM5 channels to stimulate insulin secretion from pancreatic beta‐cells. This new information confirms the hypothesis that targeting TRPM5 is a valid approach to stimulate insulin secretion and in fact, is unknowingly already widely used.

Support or Funding Information

Koenraad Philippaert is a FWO [PEGASUS]2 Marie Skłodowska‐Curie Fellow and received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska‐Curie grant agreement (665501) with the Research Foundation Flanders (FWO).
Computational and Structure‐Based Methods for the Prediction of Resistance Associated Mutations
Authors: Frey Kathleen M. , Segobia David , Boulos Monica
Published: April 2020

Drug resistance is a major limitation of therapeutic agents used for the treatment of infectious diseases and cancer. For enzyme targets, mutations in drug binding sites often lead to amino acid changes that disrupt key drug‐target interactions. In the clinical setting, these resistance‐associated mutations (RAMs) are responsible for treatment failure. In order to aid drug design efforts, it would be efficient and cost effective to develop computational and structure‐based methods to pre‐evaluate resistance mutations for enzyme drug targets during preclinical design. Our main research objective is to use computational and structure‐based methods to pre‐evaluate RAMs for enzyme drug targets. As proof of concept, we evaluated 2 enzyme targets for this study: HIV reverse transcriptase (RT), an antiretroviral target, and Glutathione S‐transferase Pi (GSTP), an anticancer target. Our computational and structure‐based methods include molecular docking, residue scanning, comparative structure analysis, and molecular dynamics (MD) simulations. Computational predictions were further validated with experimental enzyme inhibition assays. Major results indicate that this approach can predict and elucidate the effects of clinically relevant RAMs. We believe that this approach may be useful in predicting RAMs for inhibitors in development.

Support or Funding Information

Provost Seed Grant (FDU) 2019

Computer‐based methods for predicting resistance‐associated mutations

Figure 1
Arterial baroreceptors sense blood pressure through decorated aortic claws
Authors: Min Soohong
Published: April 2020

Mechanosensory neurons across physiological systems sense force using diverse terminal morphologies. Arterial baroreceptors are sensory neurons that monitor blood pressure for real‐time stabilization of cardiovascular output. Various aortic sensory terminals have been described, but those that sense blood pressure are unclear due to a lack of selective genetic tools. Here, we find that all baroreceptor neurons are marked in Piezo2‐ires‐Cre mice, and then use genetic approaches to visualize the architecture of mechanosensory endings. Cre‐guided ablation of vagal and glossopharyngeal PIEZO2 neurons eliminates the baroreceptor reflex, and aortic depressor nerve effects on blood pressure and heart rate. Genetic mapping reveals that PIEZO2 neurons form a distinctive mechanosensory structure: macroscopic claws that surround the aortic arch and exude fine end‐net endings. Other arterial sensory neurons that form flower spray terminals are dispensable for baroreception. Together, these findings provide structural insights into how blood pressure is sensed in the aortic vessel wall.

Support or Funding Information

This work was supported by NIH grants (DP1 AT009497, R01 HL132255, and OT2 OD023848) and, a Faculty Scholars Award from the Howard Hughes Medical Institute to Stephen D. Liberles. Stephen D. Liberles is an investigator of the Howard Hughes Medical Institute.

Aortic depressor nerve innervate aortic arch with distinct sensory terminals.

Figure 1

PIEZO2 neurons form aortic claws surrounding the vessel wall.

Figure 2
Arrdc4 Regulates Insulin‐Stimulated Glucose Metabolism
Authors: Dagdeviren Sezin , Shah Anu , Okawa Marinna , Melnik Veronika Y. , Sarikhani Mohsen , Foot Natalie , Kumar Sharad , Lee Richard T.
Published: April 2020

The molecular mechanisms that control glucose uptake into the cells are not yet completely defined. The Arrestin superfamily of proteins controls the inactivation, degradation and signaling of trans‐membrane receptors. Several members of the family, including Arrestin domain containing 3 and TXNIP, are now known to have important roles in metabolism. Arrestin domain containing protein 4 (ARRDC4) is a less‐investigated member of the ancient alpha‐arrestin family. Here, we show that ARRDC4 controls insulin‐stimulated glucose metabolism in vivo and in vitro. Both ARRDC4 and TXNIP suppressed insulin‐stimulated glucose uptake in differentiated adipocytes (Fig.
). In 8‐week old male C57Bl6 wildtype mice and mice with systemic deletion of the ARRDC4 gene (n=5–7/group) subjected to insulin tolerance tests (0.75U/kg), we found that mice with deletion of ARRDC4 were significantly more insulin sensitive compared to wildtype mice (Fig
). We also found that deletion of ARRDC4 in mice led to increased insulin‐stimulated Akt phosphorylation in adipose tissues (Fig
). Furthermore, in vitro and in mice, the expression of ARRDC4 is suppressed by insulin (Fig
). These data identify ARRDC4 as a regulator of mammalian insulin‐stimulated glucose metabolism.

Support or Funding Information

Supported by NIH/NIDDK to R.T.L 1R01DK107396

(A) Insulin‐stimulated H3 deoxy‐glucose uptake in differentiated 3T3‐L1 mature adipocytes that were transfected with either empty, ARRDC4 or TXNIP plasmid (n=6) (B) Insulin tolerance test (0.75U/kg) of 8‐week old ARRDC4 KO and WT mice (n=5–7) (C) Insulin‐stimulated Akt phosphorylation in adipose tissues of ARRDC4 KO and WT mice. ARRDC4 mRNA expression in the insulin‐stimulated HEPG2 cells (D) or livers of wildtype mice (E) (n=4) *p<0.05 **p<0.01.

Figure 1
SPARC: Method for Overcoming Temporal Dispersion in Unmyelinated Nerves for Imaging C Fibres with Electrical Impedance Tomography (EIT)
Authors: Tarotin Ilya , Mastitskaya Svetlana , Hannan Sana , Ravagli Enrico , Aristovich Kirill , Holder David
Published: April 2020

Fast neural EIT is a novel technique that enables imaging of evoked myelinated fibre activity in fascicles in rat sciatic nerve in vivo. However, imaging unmyelinated fibres is challenging due to temporal dispersion of the neural activity, caused by variability in fibre conduction velocities. This reduces the signal to below noise several centimetres from onset. The purpose of this work was to develop a method to overcome dispersion and allow EIT recordings of phasic activity at any distance from onset along the nerve.

Methods

Compound impedance changes (dZ) were simulated using an electrically stimulated CAPs in 1D FEM model of a C fibre
[1], with statistical analysis for 5000 fibres with conduction velocities 0.6±0.07 m/s (mean±SD)
[2]. This enabled simulation of dZ up to 20 cm from the stimulus with stimulus trains of 5/10/20/50 Hz lasting 0.25–8 s with 3 s intervals between trains (Fig.
1a). The total duration of each simulation was 100 s.

dZ was simulated 1) By coherent averaging of responses of supramaximal activity of single spikes
[3] with demodulation bandwidth of 50–300 Hz and 2) By bandpass filtering of entire evoked spike trains with bandwidths of 0.25–5 Hz which allowed significant reduction of the noise without the need for coherent spike averaging.

Random gaussian noise (0.015 μV RMS) was added to the signal to simulate realistic recording conditions. The SNR in the utilised approaches was computed for all tested stimulation and recording paradigms and compared.

Results

While coherent spike averaging was superior at short distances, its SNR fell exponentially with the distance from the onset (Fig.
1b, solid lines). The SNR with the compound demodulation was more consistent across distances (~2 at the optimal stimulation frequency of 20 Hz, Fig.
1b, dashed lines) allowing recording at ≥ 20 cm from stimulation. The estimated minimal averaging time required to obtain an SNR of 4 at 20 cm from the stimulus was equal to approximately 30 minutes at 20 Hz.

Conclusion and future work

The developed stimulation and signal processing paradigm demonstrated the feasibility of dZ recording at long distances from the stimulus. Future work is to validate it on the ex vivo crustacean leg nerve model and subsequently an in vivo mammalian model with the final objective of imaging C fibres in the vagus nerve in humans. If successful, this technique will be applied for improvement of the emerging field of bioelectronic medicines
[4].

Support or Funding Information

The work was supported by NIH SPARC grant no: 3OT2OD026545‐01S2.

(a) Example of AP and dZ responses of the C fibre subjected to repetitive 50 Hz stimuli; (b) SNR computed for the single and compound recording paradigms.

Figure 1
The Role of Coenzyme A Degradation in the Regulation of Peroxisomal Metabolism
Authors: Vickers Schuyler D. , Shumar Stephanie A. , Leonardi Roberta
Published: April 2020

Coenzyme A (CoA) is an essential metabolic cofactor that activates fatty acids and other organic acids as CoA thioesters for a multitude of metabolic pathways in different subcellular compartments. For example, CoA is utilized in the bile acid synthesis pathway and for shortening of very long chain fatty acids in the peroxisomes. The regulation of CoA levels is essential to maintain metabolic flexibility in response to changes in nutritional status and energy demands. Balance between CoA synthesis and degradation is responsible for the regulation of CoA. While CoA synthesis is controlled at the pantothenate kinase step, the intracellular degradation of CoA is controlled by Nudix hydrolases. Nudt7 is a hepatic, peroxisomal Nudix hydrolase that specifically hydrolyzes CoA species into 3’,5’‐ADP and (acyl)phosphopantetheine. Nudt7 displays a broad substrate specificity; it is active against free CoA, short‐ and medium‐chain acyl‐CoAs, including acetyl‐CoA, and the acyl‐CoA derivatives of bile acids. Nudt7 expression and activity are regulated by nutritional state, as they both decrease upon fasting. Overexpression of Nudt7 in the liver was recently found to decrease the levels of a variety of short‐chain acyl‐CoAs and choloyl‐CoA in the liver, and to decrease the rate of peroxisomal β‐oxidation. To gain further insight into the role of Nudt7 in vivo, Nudt7−/− mice were generated and their lipid metabolism assessed. Deletion of Nudt7 caused a small but significant increase in total hepatic CoA levels in the fasted state. In particular, higher concentrations of malonyl‐and acetyl‐CoA were observed. Furthermore, livers from Nudt7−/− males contained higher levels of short‐chain fatty acids and short‐chain acylcarnitines, which, combined, suggest an increase in fatty acid oxidation. In the fed state, female Nudt7−/− mice exhibited a higher concentration of selected bile acids in the intestine, while feeding of a high fat high/cholesterol diet resulted in higher bile acid levels in the liver. This increase in bile acids occurred without changes in the expression of genes involved in bile acid synthesis or transport and without a correspondent increase in the concentration of bile acid precursors 7α‐hydroxycholesterol and 7α‐hydroxycholestenone. These results suggest that higher CoA levels in the peroxisomes may mediate an increase in flux through the bile acid biosynthetic pathway by activating of the last, peroxisomal steps in the pathway.

Overall, analysis of lipid metabolism following manipulation of Nudt7 expression in mouse liver indicates that this enzyme contributes to the regulation of peroxisomal fatty acids and bile acid metabolism.

Support or Funding Information

This work was supported by West Virginia University’s School of Medicine startup foundation funding and NIH grant GM119528.
A Coupled Electromechanical Myocyte Model to Assess the Effects of 2‐deoxy‐ATP on Contractile Function in the Heart
Authors: Teitgen Abigail , McCabe Kimberly , Hock Marcus , Regnier Michael , McCulloch Andrew
Published: April 2020

Introduction

Heart failure (HF) remains a significant cause of morbidity, mortality, and medical costs. HF is characterized by reduced contractile function and inability of the heart to pump sufficient blood to meet the body’s demands. Existing treatments for HF do not permanently restore cardiac function and can lead to side effects such as impaired ventricular relaxation. 2‐deoxy‐ATP (dATP), a novel heart failure therapeutic, has been shown to increase contractile function while enhancing relaxation
[1]. Previous studies have shown that dATP increases the rate of crossbridge cycling
[2]. It has also been shown that dATP acts on the sarcoplasmic reticulum ATPase (SERCA), the pump responsible for removing calcium (Ca2+) from the cytosol, which could explain the enhanced relaxation kinetics seen experimentally
[3]. However, the mechanism of dATP is still not fully understood. Further, it is not clear whether the increased rate of crossbridge cycling with dATP treatment could aggravate energy starvation in HF.

Methods

To assess the full mechanism of dATP at the cellular level, we utilized a myocyte model containing several ion channels, including SERCA
[4]. We coupled this model to a mitochondria model to investigate whether dATP treatment aggravates energy starvation by tracking ATP, ADP, Pi, and oxygen concentrations over time
[5]. We also coupled the myocyte model to a previously developed 5 state Markov model of crossbridge cycling, which captures regulation of contraction via Ca2+ binding to troponin
[2]. The crossbridge cycling model was modified to capture the effects of sarcomere length on Ca2+ sensitivity. dATP treatment was simulated by adjusting the ATP and Ca2+ binding rates in the SERCA model and the rate of crossbridge cycling in the sarcomere model based on previous studies
[2,
3].

Results and Conclusions

We found that the combined effects of dATP on both SERCA and the sarcomere within the coupled myocyte model could explain the increase in fractional shortening and time to 50% and 90% relaxation of the Ca2+ transient seen experimentally more accurately than either model alone. Additionally, we found no evidence that dATP aggravates energy starvation, affirming dATP as a promising therapeutic for HF.

Support or Funding Information

NIH HLT32 TG
Evaluation of a Putative PPAR Modulator
Authors: Skeels Amy , Cumbay Medhane , Strom David , LaFontaine Michael
Published: April 2020

Background

Peroxisome Proliferative‐Activated Receptors (PPAR) are part of a superfamily of receptors. Of the PPARs, three forms are most prevalent, PPAR‐a, PPAR‐b/d and PPAR‐g that bind FA & FA‐derivatives to their receptor domains. Each PPAR consists of an AF‐1 domain, DNA‐binding domain, dimerization domain, and ligand‐binding domain. (1) When a lipid‐derived ligand binds to a PPAR, the PPAR heterodimerizes with a retinoid X receptor (2). This heterodimer will bind a co‐activator protein (3), which in turn activates a peroxisome proliferator response element (PPRE) (1,2). The PPRE is located in a promoter region of the target genes of a DNA sequence that will allow for trans‐activation or ‐repression of gene expression. (4.) Previous data has been collected regarding a drug compound called Compound 9, which was a dual agonist of PPARg and PPARd. The compound tested, benzyl derivative, called BMZ is a derivative of compound 9.

Objectives

Test novel compound, a demethylated derivative of a previously used compound, called BMZ, to determine its activation of PPAR receptors.

Methods

To test BMZ activation of PPAR receptors, HeLa cells were plated into 96 well plates and transfected with PPRE‐3x‐TK‐luciferase (520ng/l), Trans IT‐X2 transfection reagent, and serum reduced media. The cells were then treated with troglitazone, pioglitazone, and BMZ with the following concentration in separate wells in triplicate, 0.1mM, 0.3 mM, 1.0 mM, 3.0 mM, 10 mM, 30 mM. Triplicate wells were also treated with 10 mM of BMZ with 10 mM of troglitazone. Finally, a separate triplicate of Compound 9 was added. A luciferase assay was conducted 24 hours after application of drug in five, five minute intervals.

Results

The results showed that there was some activation of PPAR receptors for the novel BMZ compound. Furthermore, when BMZ was added to troglitazone, there was an decreased activation of the receptors.

Support or Funding Information

Marian University College of Osteopathic Medicine Internal Grant

Relative Luminescence Units of various PPAR‐gamma agonists

Figure 1
Opioids Cause Vascular Remodeling via Changes in Cofilin‐ERK Signaling: Female Mice Present Higher Risk of Developing Morphine‐induced Cardiovascular Disease than Male Mice
Authors: Cheon Soyoung , Tomcho Jeremy C. , Bearss Nicole R. , Joe Bina , McCarthy Cameron G. , Wenceslau Camilla F.
Published: April 2020

According to the most recent CDC Annual Surveillance Report of Drug Related Risks and Outcomes, more than 47,600 people died from an opioid overdose in 2017 in the United States. The magnitude of the national crisis raises questions on intersecting points between opioid abuse and the main systems of the human body. Recent statistical analyses have shown that chronic use of prescription or illicit opioids leads to an increased risk of a cardiovascular event, which was higher among women than men. We hypothesized that chronic exposure to exogenous opioids causes sex‐specific vascular remodeling via changes in actin polymerization, leading to an increased risk of cardiovascular disease. In a preliminary study to test our hypothesis, we observed that treatment with the exogenous opioid morphine (≥ 24 hours) induced vascular smooth muscle cell proliferation. Acknowledging the emerging roles of cofilins and extracellular signal‐regulated kinases (ERK) in the regulation of cell proliferation by mediating actin dynamics, we investigated the effects of morphine (MOR) on the intracellular levels of both proteins in vasculature. Mesenteric resistance arteries (MRA) from 10 week old wild type C57BL/6NTac male and female mice were extracted (n=4). The MRA of each animal were isolated and divided equally into control and treatment groups. The control and treated samples were incubated in DMEM with 1% penicillin‐streptomycin at 37°C for 24 hours in the absence or presence of MOR (1μM) respectively. We observed that MOR increased phosphorylated/inactivated cofilin 1.6 fold [arbitrary units (AU), control: 0.59 ± 0.14 vs MOR: 0.95 ± 0.10*, *p<0.05] and increased phosphorylated/activated ERK 1.8 fold (AU, control: 0.51 ± 0.11 vs MOR: 0.90 ± 0.13*, *p<0.05) in the MRA from female mice. On the other hand, MOR decreased phosphorylated ERK 1.5 fold (AU, control: 0.91 ± 0.08 vs MOR: 0.59 ± 0.11*, *p<0.05) but had no effect on the levels of phosphorylated cofilin (p>0.05) in the MRA from male mice. Our data suggest that exogenous opioids change actin dynamics in arteries from both male and female mice. Further, this study is the first to demonstrate that prolonged exposure to morphine may increase cardiovascular risk in females when compared to males.

Support or Funding Information

This work was supported by National Institutes of Health (NIH) (NIH: R00GM118885).

Levels of phosphorylated cofilin (left) and ERK (right) in MRA from male and female mice after 24 hours of treatment with morphine (MOR, 1μM) or vehicle.

Figure 1
Mitochondria Influence NMDA Receptor‐mediated Currents in Hypothalamic Magnocellular Neurons: Altered Mechanisms during Heart Failure
Authors: Ferreira-Neto Hildebrando Candido , Stern Javier
Published: April 2020

Hypothalamic magnocellular neurosecretory neurons (MNNs) located in the supraoptic (SON) and paraventricular nuclei (PVN) play a critical role in the pathophysiology of heart failure (HF). Mitochondria (MITO) plays an important role in the regulation of intracellular Ca2+ dynamics, and several studies observed that morphological and functional MITO changes contribute to the development and maintenance of various brain diseases. We recently demonstrated that NMDA receptors activation triggers a Ca2+−dependent stimulation of SK channel in MNNs from Sham rats, and that this coupling acts as a negative feedback mechanism restraining the magnitude of the NMDA receptor‐mediated current (INMDAR) and its excitatory effects. Importantly, we found that the NMDA‐SK channel coupling is blunted in MNNs from HF rats. Still, whether MITO regulation of intracellular Ca2+ dynamics influence INMDAR and whether this effect is altered during HF is unknown. To address this question, we obtained whole‐cell patch clamp recordings from MNNs (holding potential: −50 mV) in slices from Sham and HF rats. We found that focal application of NMDA (50 μM, 300 ms) evoked an INMDAR whose magnitude was similar in Sham and HF rats (p>0.05), as previously reported. Application of CCCP (10 μM, 5 s), a mitochondrial uncoupler, significantly increased the evoked INMDAR in MNNs from Sham rats (p<0.05), but had no effect in MNNs from HF rats (p>0.05). Intracellular dialysis of MNNs with the Ca2+ chelator BAPTA (10 mM) significantly augmented INMDAR in MNNs from Sham rats compared to the control condition (p<0.05), and occluded the CCCP‐induced INMDAR increase. Taken together, our data supports that mitochondria regulate, in a Ca2+‐dependent manner, the magnitude of INMDAR in MNNs from Sham rats, a phenomenon that is blunted during HF.

Support or Funding Information

R01HL090948
Deletion of the mechanistic‐target‐of‐rapamycin (mTOR) from collecting duct principal associated with increased ubiquitinylation of epithelial sodium channel (ENaC)
Authors: Chen Bruce , Fluitt Maurice B. , Shivapurkar Narayan , Brown Aaron , Ecelbarger Carolyn Mary
Published: April 2020

The mechanistic target of rapamycin (mTOR), a serine‐threonine‐specific kinase, is a cellular energy sensor, integrating growth factor and nutrient signaling. In the renal collecting duct (CD) or in related cell cultures, mTOR has been demonstrated to upregulate activity of the epithelial sodium channel (ENaC). Our aim was to determine the impact of CD‐principal‐cell‐select mTOR deletion on the adaptations to changes in dietary sodium and elucidate ENaC regulation. Male and female CD‐PC mTOR KO mice and wild‐type (WT) littermates were generated by Cre‐lox recombination using aquaporin‐2 (AQP2) promoter to drive Cre‐recombinase. KO mice had 20–40% reduced protein levels of all three subunits of ENaC under low‐ (0.5% Na+, LSD) or normal‐NaCl (< 0.02% Na+, NSD). Switching from NSD to LSD resulted in transiently increased urine sodium losses (day 1) in the KO, with adaptation by day 2. Likewise, KO were significantly impaired (20% less Na+ excretion) on day 1 in transitioning from NSD to high‐NaCl (4%) diet. Under NSD, an in vivo acute test of benzamil‐sensitivity supported reduced (~30%) ENaC activity in the KO; however, 24‐hour urine collection after dietary BNZ (5 mg kg·bw) showed increased Na+ losses in the KO. No genotype differences in blood pressure (BP, tail cuff) were observed under NSD; however, LSD surprisingly increased BP in WT, but not KO. Apical targeting of ENaC subunits with LSD appeared intact (after 7‐days of LSD). To interrogate the mechanisms for reduced ENaC subunit protein levels, an ubiquitination ELISA assay was conducted on cortex homogenates probing for ubiquitinated a‐ENaC. KO mice demonstrated 50–100% higher density for immunoprecipitated ubiquitin probed for a‐ENaC, as compared to WT mice in both males and females. Our data support a role for mTOR in ENaC activity and sodium homeostasis potentially via regulation of ENaC subunit turnover.

Support or Funding Information

BGRO Georgetown University and Marriott Cardiovascular Foundation (CE); American Diabetes Association (MF); Department of Biochemistry, GU (AB)

ENaC was localized to the apical membrane in both WT and KO mice with low‐NaCl feeding.

Figure 1

Increased ubiquitination of alpha‐ENaC in CD‐principal cell targeted mTOR KO mice

Figure 2