bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Inducible Nitric Oxide Synthase (iNOS) by Cyclin‐Dependent Kinase 7 (CDK7) in Endothelial Cells
Authors: Shetty Shruthi , Spaulding Amelia M. , Baker Cassadie , Baine Stephen H. , Hoyt Dale G.
Published: April 2020

E. coli endotoxin (LPS) combined with interferon‐γ (IFN) induces inducible nitric oxide synthase (iNOS), with production of nitric oxide (NO) and reactive nitrogen species in endothelial cells. Appropriate manipulation of iNOS during onset or resolution of inflammation may be useful. Since transcription is required for induction of iNOS by LPS/IFN, we hypothesized that depletion of cyclin dependent kinase (CDK) 7, a promoter of transcription, would antagonize induction. Mouse aortic endothelial cells (MAEC) were transfected with Non‐targeted (NT) control RNA or CDK7 siRNA to deplete the kinase, followed by stimulation with LPS/IFN for 12 h. Rather than suppressing, CDK7 depletion increased induction of iNOS, and its dimerization and NO production. The effect was post‐transcriptional in that mRNA induction was unaffected, while iNOS protein turnover was reduced in the absence of CDK7. Cellular calpain activity, which is required for iNOS protein degradation in MAEC, was unaffected by CDK7 depletion, suggesting an effect on iNOS itself. Indeed, iNOS in lysates of kinase‐deficient cells was resistant to in vitro digestion with exogenous calpain. In conjunction with protease resistance, the association of iNOS with calmodulin, known to inhibit its digestion by calpain, was increased in cells lacking CDK7. These results suggest that CDK7 normally restrains iNOS induction by limiting calmodulin binding and maintaining calpain‐mediated turnover. By regulating iNOS enzyme complexes, CDK7 may serve as a novel target for manipulation of vascular NO.

Support or Funding Information

Support was received from the Popat Patil Fellowship (SS).
Role of Bronchopulmonary C‐fibers in Bleomycin‐induced Rapid Shallow Breathing (RSB) and Idiopathic Pulmonary Fibrosis (IPF) in Mice
Authors: Wei Wan , Zhuang Jianguo , Gao Xiuping , Jiao Yang , Xu Fadi
Published: April 2020

IPF is characterized by an irreversible loss of lung function associated with RSB, dyspnea, and hypoxemia, which often leads to death within 3–5 years due to respiratory failure. Inflammation and abnormal wound healing of the interstitial and alveolar spaces in the lungs with excessive proliferation of myofibroblasts (over‐secretion/expression of collagens and α‐smooth muscle actin, α‐SMA) result in fibrosis. Substance P (SP) is known to participate in fibrosis in multiple organs, such as cardiac, renal and liver fibrosis, and wound healing process, but its role in pathogenesis of IPF is unexplored. Because inflammation mediators could stimulate bronchopulmonary C‐fibers (PCFs) to release SP (the major source of inflammatory SP releasing in the lungs) and evoke neurogenic inflammation and RSB (dyspnea), here we asked whether PCFs and neurogenic SP would involve in the pathogenesis of IPF. To this end, four groups of mice pretreated with neonatal capsaicin (for PCF degeneration) or its vehicle received endotracheally (by transoral route) saline (Ctrl) or bleomycin (BLM) respectively. On the 21st day post pretreatment, ventilation, heart rate, and SpO2 were measured. After subsequent euthanasia, BALF and the right lung tissues were collected to detect inflammatory cells, SP, and hydroxyproline, while the left lungs were fixed for histological assessment of IPF, pulmonary inflammation, and α‐SMA immunoreactivity. The results showed that in PCF intact mice, BLM induced RSB, hypoxemia, IPF (upregulation of pulmonary SP, α‐SMA, and hydroxyproline), and pulmonary inflammation. PCF degeneration: 1) eliminated the BLM‐induced RSB and SP upregulation; 2) failed to affect the BLM‐induced hypoxemia; and 3) aggravated IPF (upregulation of α‐SMA and hydroxyproline) and pulmonary inflammation. Our results suggest that PCFs are responsible for the BLM‐induced RSB. On the other hand, PCFs, likely via releasing SP, are protective for the lungs from genesis of IPF through suppressing pulmonary inflammation, α‐SMA and hydroxyproline.
Impact of Short, Long, and Intermittent Access Conditions on MDPV and Cocaine Self‐administration in Male and Female Rats
Authors: Doyle Michelle R. , Sulima Agnieszka , Rice Kenner C. , Collins Gregory T.
Published: April 2020

Despite decades of research, the behavioral, pharmacological, and neurobiological determinants of one’s vulnerability to develop a substance use disorder are not well understood. When rats are allowed to self‐administer cocaine under short‐access conditions, patterns of drug intake tend to be well‐regulated; however, manipulating drug access conditions (e.g., long‐access and intermittent‐access) can produce neurobiological and behavioral changes thought to be related to addiction. In contrast to cocaine, when rats self‐administer the synthetic cathinone 3,4‐methylenedioxypyrovalerone (MDPV) under short access conditions, a subset display high levels of dysregulated drug‐taking behavior. However, MDPV has not been studied under extended access conditions, and it is unknown whether this MDPV phenotype is simply an earlier manifestation of the addiction‐like phenotype observed with cocaine, or whether the two phenotypes are qualitatively different. The current studies used male and female Sprague Dawley rats self‐administering MDPV (0.032 mg/kg/infusion) or cocaine (0.32 mg/kg/infusion) to test the hypotheses that (1) under short‐access conditions, rats self‐administering MDPV exhibit higher levels of drug taking, greater levels of responding when drug is not available, and reduced sensitivity to punishment by foot shock (i.e., addiction‐like behaviors); and (2) that 21 days of long‐ and intermittent‐access to MDPV or cocaine self‐administration will result in greater addiction‐like behaviors compared to short‐access self‐administration. After short‐access self‐administration, rats that self‐administer MDPV exhibit greater addiction‐related behaviors than rats self‐administering cocaine, as measured by the three criteria detailed above. Though long‐and intermittent‐access produce some changes in patterns of drug‐taking behavior, the access condition manipulation does not appear to systematically alter the addiction‐like behaviors in either rats self‐administering cocaine or MDPV. These results suggest that the phenotype displayed by rats self‐administering MDPV is not the same phenotype as that exhibited by rats with a history of self‐administering cocaine. Therefore, MDPV self‐administration may be a useful method to study individual differences in drug‐taking behavior under short‐access conditions.

Support or Funding Information

This work was supported by NIH/NIDA (R01 DA039146; GTC) and NIDA‐ and NIAAA‐IRPs (KCR).
The Therapeutic Potential of Apoptotic Mesenchymal Stem Cells in Muscle Regeneration
Authors: Huang Wenbai , Suggs Laura J. , Farrar Roger P.
Published: April 2020

Macrophages are critical for muscle regeneration and have an unusually high level of responsiveness to their environment. Because of this sensitivity, their beneficial effects may be realized only upon certain activation conditions. Previous studies in our lab have shown that transplantation of classically activated macrophages and anti‐inflammatory macrophages sorted from injured muscle enhanced functional regeneration in ischemia‐reperfusion muscles. The phagocytosis of apoptotic cells that are present in injured muscle or from transplantation serves as a well‐known activation signal for macrophages to initiate a regenerative and anti‐inflammatory profile leading to enhanced muscle regeneration. The aim of this study is to investigate if the phagocytosis of apoptotic mesenchymal stem cells (MSCs) can polarize bone marrow‐derived macrophages (BMDMs) towards a regenerative and anti‐inflammatory phenotype and subsequently if apoptotic MSCs in different stages of apoptosis have differential effects. We induced apoptosis in MSCs and incubated them at different time intervals to obtain differential stages of apoptosis, which were characterized by the amount of externalized phosphatidylserine (PS) on their cell membrane. Apoptotic MSCs with different concentrations of PS were then co‐cultured with BMDMs for 48 hours. Cell surface markers and gene expression of characteristic of pro‐ and anti‐inflammatory polarized BMDMs were measured. Our results showed that apoptotic MSCs without incubation significantly increased the expression of F4/80 and CD206 on the cell surface of BMDMs. They also increased the gene expression of MRC‐1 and IL‐10 as well as decreased the expression of iNOS and IL‐1β in co‐cultured BMDMs. The extent of these effects exhibited a decreasing trend when incubation time of apoptotic MSCs increased. In conclusion, the extent of MSC apoptosis as characterized by PS expression resulted in differential anti‐inflammatory and regenerative effects on BMDMs, wherein the apoptotic MSCs in the earlier stage of apoptosis result in the greatest increase in anti‐inflammatory phenotype, which reveals their therapeutic potential in muscle regeneration.

Support or Funding Information

This project is funded by National Institutes of Health Grant 2R01EB015007‐05A1 (to Laura J. Suggs and Roger P. Farrar)
Anticancer Activity of Curcumin and Curcumin Degradation Products in Combination with Silibinin
Authors: Sayyed Alhan , Ezekiel Uthayashanker
Published: April 2020

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in men and women in the United States. Several studies have shown that a diet high in fruits, vegetables, and spices is strongly associated with reduced risk of developing colorectal cancer. Fruits and vegetables contain a wide variety of phytochemicals that offer protection against colorectal cancer. The anticancer activity of healthy diets may be attributable to a synergy among various phytochemicals present in the diet. We have previously shown that phytochemicals derived from turmeric (e.g. curcumin) and milk thistle (e.g. silibinin) have exhibited synergistic anticancer activity against colorectal cancer cells. In the present study we hypothesize that the curcumin degradation products, tran‐ferulic acid, vanillylidenacetone, ferulic aldehyde, and vanillin in combination with silibinin will exhibit synergistic anticancer activity. Our results indicate that the degradation products of curcumin plus silibinin did not exhibit synergistic anticancer activity. We found that only curcumin plus silibinin exhibited synergistic anticancer activity.

Support or Funding Information

AS is supported by the DeNardo Education and Research Foundation grant.
Science Communication Boot Camp: An American Association for Anatomy program to empower members with skills in effective public communication strategies
Authors: Organ Jason , Hoffmann-Longtin Krista
Published: April 2020

As scientific fields have become more complex in the 21st century, some Americans have given up trying to understand what we do, leading to science illiteracy. Although scientists have reached consensus on many issues, such as climate change accelerated by humans, and evolution, the public and (American) elected officials have not always accepted this consensus. According to the Pew Center, there is a wide gap between scientists’ and the public’s perception of how science is used to inform government policy. Without expert voices communicating science to the public and policy‐makers, fields like healthcare, climate science, and evolutionary biology—all fields that are inextricably linked to anatomy—have been overrun with conspiracy theories and misinformation.

The timing is right to make science communication a fundamental part of doing science. And to do that, we must train our students/faculty in the skills necessary to be effective communicators. Over the last several years, the American Association for Anatomy (AAA) has prioritized professional development in science communication for researchers, educators, and practitioners at its annual meeting. Furthermore, AAA recently adopted a new strategic plan which brings science communication front and center, where the public is engaged through the promotion of anatomical sciences. To support this new science communication initiative, AAA hosted its inaugural Science Communication Boot Camp (SCBC) on the campus of Indiana University School of Medicine in Indianapolis in July 2019. This three‐day program brought participants together to learn effective communication skills from the faculty of the Alan Alda Center for Communicating Science and from representatives of the Federation of American Societies for Experimental Biology science policy staff. This talk reports on the SCBC program and demonstrates some of the hands‐on activities employed.

Support or Funding Information

This study was granted exempt status by the Institutional Review Board of Indiana University School of Medicine. Funding was provided by the AAA Innovations Grant Program and the Indiana University School of Medicine Office of Faculty Affairs, Professional Development, and Diversity.
Comparison of Wire and Disc Leads to Activate the Inspiratory Muscles in Dogs
Authors: Kowalski Krzysztof E. , DiMarco Anthony F.
Published: April 2020

Objective

HF‐SCS is a unique method of inspiratory muscle activation which results in coincident activation of the diaphragm and inspiratory intercostal muscles via spinal cord pathways and has the potential to provide respiratory support in ventilator dependent persons with spinal cord injury (SCI). The purpose of the present study was to examine the effectiveness of wire leads, which can be placed using minimally invasive techniques, to activate the inspiratory muscles.

Methods

In 5 anesthetized dogs, in separate trials, disc and wire leads were inserted onto the ventral epidural space at the T1–T4 spinal cord levels. The effects of electrical stimulation with disc, single wire, and two wire leads placed in parallel were compared.

Outcome measures

Airway pressure generation following stimulation with disc and various configurations of wire leads were compared.

Results

Several different configurations of wire leads resulted in airway pressures that were similar to those generated with monopolar stimulation with disc leads (D). For example, monopolar stimulation (3mA, 300Hz, 0.2ms) with parallel wire leads in which each of the electrode contacts at the T2 were connected together and functioned as a common cathode (Y‐connection) and monopolar stimulation (separate cathode) at T2 both with a remote ground resulted in airway pressures that were 91±6 and 92±4%, respectively, of those achieved with D (NS for both). Bipolar stimulation (3mA, 300Hz, 0.2ms) with parallel wire leads using Y‐connection and with a separate cathode at the T2 and T3 level resulted in airway pressures that were 96 ± 4 and 94 ± 4%, respectively, of the pressures achieved with D (NS for both).

Conclusion

These results suggest that specific configurations of wire leads, which can be placed via minimally invasive techniques, result in comparable activation of the inspiratory muscles compared to disc leads and may be a useful technique to restore ventilatory support in persons with SCI.

Support or Funding Information

Support

NIH‐NINDS (R01NS064157)

Disclosure

Dr. DiMarco is a founder of and has a significant financial interest in Synapse BioMedical, a manufacturer of diaphragm pacing systems and holds patents for spinal cord stimulation to restore cough and respiration (5,678,535; 5,911,218; 5,999,855; 8,751,004). Drs. DiMarco and Kowalski hold the U.S. patents for technology related to the content of this manuscript: Respiratory Muscle Activation by Spinal Cord Stimulation (8,352,036).
Impaired Mitochondrial Respiration in Hermansky‐Pudlak Syndrome 1‐Defective Alveolar Type 2 Cells is Associated with Enhanced Mitochondrial Fission
Authors: Kook Seunghyi , Meng Shufang , Rasmussen Megan L. , Trenary Irina , Young Jamey D. , Gama Vivian , Guttentag Susan H.
Published: April 2020

Alveolar epithelial injury is central to the pathogenesis of pulmonary fibrosis in lung diseases across the lifespan, from bronchopulmonary dysplasia and childhood interstitial lung disease to idiopathic pulmonary fibrosis. Disturbances in mitochondrial structure that alter function have been associated with lung epithelial cell injury, yet molecular mechanisms regulating mitochondrial dynamics in the setting of pulmonary fibrosis remain unclear. Hermansky‐Pudlak Syndrome (HPS) patients, especially those with mutations in HPS1 and HPS4, develop fibrosis in earlier age than patients with idiopathic pulmonary fibrosis (IPF), suggesting that loss of HPS gene function drives alveolar epithelial injury. Here we show enhanced mitochondrial fragmentation in HPS1‐defective alveolar type 2 (AT2) cells associated with impaired phosphorylation of Dynamin‐related protein 1 (DRP1) at Serine (Ser)637 (inactivating phosphorylation). Ectopic expression of constitutively active GTPase Rab32, Rab32(Q85L), designed to bypass the guanine nucleotide exchange factor (GEF) function of HPS1‐HPS4 complex (BLOC‐3), in MLE15/DHPS1 cells restores mitochondrial architecture and increases p‐Ser637‐Drp1. In addition, MLE15/DHPS1 cells exhibit markedly reduced mitochondrial respiration, signified by reduced oxygen consumption (OCR), and robust glycolysis, indicated by a brisk increase in extracellular acidification rate (ECAR) after addition of glucose and accumulation of lactate in culture media. Despite robust glycolysis, MLE15/DHPS1 cells have lower total cellular ATP than MLE15/WT cells. Moreover, MLE15/DHPS1 cells demonstrate dependency on glucose as a nutrient source, reflected in activation of the energy sensor 5’ AMP‐activated protein kinase (AMPK) and cell death upon decreasing glucose in culture media. Taken together, our results suggest that HPS1, functioning in a heterodimer with HPS4 as an activator of Rab32, plays a central role in balancing mitochondrial fission/fusion in AT2 cells, while loss of HPS1 disrupts oxidative phosphorylation and drives metabolic reprogramming in AT2 cells.

Support or Funding Information

NIH (GM108807/SG). SG is the Julia Carell Stadler Professor of Pediatrics. NIH/NIGMS 1R35GM128915‐01 (to VG); NIH/NCI 1R21 CA227483‐01A1 (to VG). NIH/R01 DK106348 and U01 CA235508 (to JY)
Examination of the demyelination process in mGFAP‐driven conditional transient receptor potential ankyrin 1 (TRPA1) receptor knockout mice
Authors: Pintér Erika , Böcskei Kata , Kriszta Gábor , Sándor Zoltán , Nemes Balázs , Ács Péter , Komoly Sámuel , Berente Zoltán
Published: April 2020

Transient receptor potential ankyrin 1 (TRPA1) receptors are non‐selective cation channels responsive to a variety of exogenous irritants and endogenous stimuli including products of oxidative stress. It is mainly expressed by primary sensory neurons however, expression of TRPA1 by astrocytes and oligodendrocytes has recently been detected in the mouse brain. Genetic deletion of TRPA1 was shown to attenuate cuprizone‐induced oligodendrocyte apoptosis and myelin loss in mice. In the present study we investigated the astrocyte‐specific mGFAP‐driven conditional TRPA1 gene deficient mice in the cuprizone model. Astrocyte‐specific knockout animals were generated by crossbreeding GFAP‐Cre+/− and floxed TRPA1 (TRPA1Fl/Fl) mice. Cuprizone was administered during 6 weeks and demyelination was followed by magnetic resonance imaging (MRI). At the end of the treatment, myelin loss and glial activation were also investigated by histological methods. The results of the MRI showed significantly increased signal intensity in cuprizone‐treated GFAP‐Cre−/− TRPA1Fl/Fl mice compared to GFAP‐Cre+/− TRPA1Fl/− and GFAP‐Cre+/− TRPA1Fl/Fl animals on week 3 and week 4. However, by week 6 of the treatment the difference was not detectable by either the MRI or the histological methods. According to the currently available data, we presume that TRPA1 receptors localized on astrocytes can be activated by reactive oxygen species in response to cuprizone challenge and the consequent release of pro‐inflammatory mediators contributes to the progression of oligodendrocyte apoptosis. However, the global TRPA1 KO mice presented a more attenuated demyelination compared to the conditional KO mice, it could be hypothesized that TRPA1 receptors expressed by other cell types (neurons, oligodendrocytes) in the brain may also participate in the demyelination process.

Support or Funding Information

This research was funded by grants “The role of neuro‐inflammation in neurodegeneration: from molecules to clinics” (EFOP‐3.6.2‐16‐2017‐00008); Hungarian Brain Research Program 2. 2017‐1.2.1‐NKP‐2017‐00002. and the University of Pécs is acknowledged for a support by the 17886‐4/23018/FEKUTSTRAT excellence grant. G.K. was supported by Richter Gedeon Centenárium Foundation.
Assessing the Global Impact of an Adenylate Kinase Isoenzyme on AMPK and mTOR Signaling Pathways Through Metabolic Profiling in both Brain and Lung Cancer Cell Lines
Authors: Salladay-Perez Ivan Alexander , Lanning Nathan J.
Published: April 2020

In a variety of human cancers, it is well observed that specific altered bioenergetic programs must be sustained in order to maintain the cancer cell phenotype. Well‐described genes play a role in sustaining energy flux for the cancer cell and recent studies add the regulation of AK4 expression, an adenylate kinase localized to the mitochondrial matrix, to the list. Our results demonstrate that in response to altered AK4 expression in cancer cells, intracellular ATP levels drastically increase. Secondly, we demonstrate that in response to perturbed AK4 expression in cancer cells, two critical homeostatic signaling pathways, the 5′ AMP‐activated protein kinase (AMPK) and a mammalian target of rapamycin (mTOR) pathways, are significantly altered. Both mTOR and AMPK proteins govern a metabolic pathway in which they sense cellular energy levels and nutrient status in response to regulate cancer cell growth and/or proliferation. Lastly, AK4 also has an impact on other bioenergetic pathways. For instance, AK4 can catalyze the reaction GTP + AMP ↔ GDP + ADP24 and sustain critical energy flux of nucleotides in the cell. Because AK4 expression appears to regulate cellular metabolic responses at multiple levels, further analyses by obtaining a metabolomic profile is necessary to highlight the global impact AK4 exerts on all the metabolites in the cancer cell. To do this, cell lines will be engineered without the AK4 gene in glioblastomas, lung adenocarcinomas, and HeLa cells then analyzed to identify all known metabolites in the system. It is hypothesized that without the expression of an AK4 protein there will be indirect or direct disruptions in the global nucleotide pools by either increasing nucleotide biosynthesis or decreasing nucleotide consumption. The outcome of this study will add to the small library of research on AK4 as well as provide a comprehensive understanding on the enzymatic activity and interactions where AK4 exerts its biological effects on cancer cell metabolism.

Support or Funding Information

Funded by National Institutes of Health (NIH) National Institute of General Medical Sciences (NIGMS) Grant 1R15GM123382‐01.
Rapid Real‐Time Ubiquitination Assays as Alternatives to Western Blotting
Authors: de Silva Ruvindi , Page Richard C.
Published: April 2020

The refolding and degradative pathways of the protein quality control system converge through the chaperoned ubiquitination complex formed by the interaction of the E3 ubiquitin ligase CHIP (
C‐terminus of
Hsp70
Interacting
Protein) and the ATP‐dependent chaperone Hsp70 (70 kilodalton heat shock protein). We have applied the CHIP/Hsp70 chaperoned ubiquitination system as a test for our newly developed ubiquitination assay, presenting a viable alternative to traditional western blots for detection of ubiquitination. Here we describe the cutting‐edge biolayer interferometry ubiquitination assay to rapidly assay ubiquitination in real time. Our BLI ubiquitination assay is recorded with 1‐second resolution and is the first assay to allow for determination of rate and extent of ubiquitination in real‐time. This assay detects formation of polyubiquitin chains directly on a biosensor‐bound target. Polyubiquitination of substrates using wild‐type components produces a robust signal. Reagent concentrations and quantities are similar to those used by traditional western blot‐based ubiquitination assays. Results are returned instantaneously and result in substantial time savings.

Support or Funding Information

NIGMS R35 GM128595
Characterization of L382 and S383 in the βDELSEED‐motif of ATP synthase
Authors: Steiner Amanda Jayne , Ahmad Zulfiqar
Published: April 2020

Background

Antibiotic resistance is rising on an alarming rate. Finding alternative ways to combat microbial infections is a matter of human survival. Selective inhibition of microbial ATP synthase has the potential to eradicate antibiotic resistant microbes. ATP synthase is the chief source of ATP production in almost all organisms. Antimicrobial properties of some venom peptides are connected to the binding and inhibition of ATP synthase. Peptides bind at the bDELSEED‐motif of E. coli ATP synthase and inhibit Escherichia coli ATP synthase to variable degrees. In order to understand the selective binding and inhibition of microbial ATP synthase it is essential to fully elucidate the binding site. Currently our lab is studying the role of Leu‐382 and Ser‐383 of the bDELSEED‐motif in peptide binding.

Method

Growth properties of wild type, null, and bDELSEED‐motif mutant E. coli strains were checked on minimal media with 3% glucose media and succinate media with 1/4th case amino acids. Wild type and mutant membrane bound F1Fo enzymes were isolated by growing on minimal media to late log phase. Cells were harvested in super centrifuge, French Pressed, and finally membrane‐bound ATP synthase isolated by ultracentrifugation. Purity of isolated protein was validated by western blot using anti‐F1‐β antibody. Biochemical inhibition of wild type and mutant ATP synthase was validated by NBD‐Cl. βLeu‐382 and βSer‐383 mutants were inhibited by honey bee venom peptide melittin. Time kinetics for full enzyme inhibition was determined along with reversibility of enzyme inhibition.

Results

Our growth results show that mutant strains grew between wild type and null strain on limiting glucose. Growth on succinate media corroborate the growth on limiting glucose. Melittin inhibited mutant enzymes to variable degrees. Moreover, we found that loss of βLeu‐382 and βSer‐383 residues caused reduced level of peptide binding at βDELSEED‐motif.

Conclusions

We conclude that residue Leu‐382 and Ser‐383 of βDELSEED‐motif are essential for proper orientation and binding of peptides at the peptide‐binding site. Moreover, βLeu‐382 and βSer‐383 play important role in maintenance of the βDELSEED‐motif.

Support or Funding Information

This work was supported by the KCOM Biomedical Sciences Graduate Program Grant Award Fund # 851‐044 to AS and ZA.
Structural and Functional Characterization of Fumarase C from Salmonella enterica
Authors: Maki Ryan R. , Winga Eden R. , Bhattacharyya Basudeb R. , May John F.
Published: April 2020

The citric acid cycle is a vital pathway for virulence of facultative bacterial pathogens. Two of these bacteria, Salmonella enterica and Escherichia coli, pose a major threat for foodborne illness. Here we present the characterization of an enzyme in the citric acid cycle as targets for the development of novel antibacterial agents. Fumarase C catalyzes the reversible reaction of fumarate to malate. Fumarate conversion to malate in a necessary piece of the CAC cycle. S. enterica has been shown to lose virulence when the genes encoding its fumarase C enzyme (SeFumC) are knocked out, making this protein a potential drug target against S. enterica infections. To gain kinetic and structural knowledge of SeFumC, we have cloned the gene encoding FumC into a plasmid for overexpression. We have purified SeFumC‐His6 using immobilized metal affinity chromatography. We have baseline values of Michaelis‐Menten kinetic parameters for the SeFumC. We are investigating the stability of SeFumC folding using circular dichroism. To facilitate structural analysis, we have identified a condition that promotes crystallization of SeFumC. Characterizing the kinetic and structural features of SeFumC will aid in the potential development of future antibiotics that target the citric acid cycle in bacterial pathogens.

Support or Funding Information

UWL Eagle Scholars Apprenticeship Program
B CELLS CONTRIBUTE TO HYPERTENSION AND CYTOLYTIC NK CELL ACTIVATION IN RESPONSE TO PLACENTAL ISCHEMIA OF PREGNANCY
Authors: Herrock Owen T. , Amaral Lorena , Fitzgerald Sarah , Lemon James , Deer Evangeline , Ibrahim Tarek , Cambpell Nathan , Usry Nathan , LaMarca Babbette
Published: April 2020

Preeclampsia (PE), new onset hypertension during pregnancy, is associated with a pro‐inflammatory state and B lymphocytes secreting agonistic autoantibodies against the angiotensin II type 1 receptor (AT1‐AA). Two recognized subsets of B Cells are B1 and B2 B Cells. B2 B Cells are the classical B cell involved in adaptive immune responses and production of antibodies through affinity maturation. B1 B Cells produce natural antibodies and have recently been shown to communicate through TH17 cells, and are implicated in the pathophysiology of PE through AT1‐AA. The Reduced Uterine Perfusion Pressure (RUPP) rat model of placental ischemia recapitulates many characteristics of PE. We have previously shown that either B cell depletion or AT1‐AA epitope binding protein (7AA) in RUPP rats improved hypertension and AT1‐AA activity. The AT1‐AA is a specific IgG3 antibody to the AT1 receptor and thus could be produced by B1 or B2 B Cells in PE. This information would add to the design of new therapies targeting one B cell type for depletion. Therefore, we hypothesize that B cells and specifically B2 cells cause hypertension and NK cell activation during pregnancy through production of the AT1‐AA. To test this hypothesis, total splenic B Cells and B2 cells were isolated from RUPP rats on Gestation Day (GD)19 RUPP and adoptively transferred into NP rats on GD12, blood pressure (MAP), placental and circulating cNKs were measured by flow cytometry on GD19. To test the role of AT1‐AA in the hypertension, 7AA was administered to a group of recipient rats on GD14. MAP increased in NP+RUPP B cells to 117 ± 1 mmHg, n=10, and to 111 mmHg ± 3, n=8, in NP+RUPP B2 cells, compared to NP rats(p<0.001). Administration of 7AA attenuated hypertension in response to total RUPP B cells (99 mmHg ± 6, n=5)(p<0.01) and in response to RUPP B2s (95.5mmHg ± 4.5, n=2). Circulating cNKs were elevated in NP+RUPPBcells (6 ± 2.5, n=5)(p<0.01) and in NP+RUPP B2 cells (8 ± 3.33, n=4)(p<0.01) compared to NP (0.67 ± 0.33, n=10). Placental cNKs were elevated in NP+RUPPBcells (2.32 ± 0.73, n=6)(p<0.01) and in NP+RUPP B2 cells (1.33 ± 0.24, n=3)(p<0.01) compared to NP (0.2250 ± 0.133, n=10). Based on these data, we conclude that total B Cells and the B2 cell subset activated in response to placental ischemia cause hypertension via secretion of the AT1‐AA and NK cell activation during pregnancy and thus indeed play an important role in PE. Future studies will examine the role of B1 cells and their interaction with TH17s as possible contributors to AT1‐AA and hypertension during PE and in response to RUPP total B cells and RUPP B2 B cells.

Support or Funding Information

R01HD067541‐06, P20GM121334, AHA 19CDA34670055, T32HL105324
Expression Levels of p21‐activated kinase (PAK1) in the Skeletal Muscle Exert Feedback to Influence Pancreatic β‐cell Functionality in vivo
Authors: Merz Karla E. , Ahn Miwon , Tunduguru Ragadeepthi , Salunkhe Vishal A. , Olson Erika M. , Oh Eunjin , Veluthakal Rajakrishnan , Yoder Stephanie M. , Zhang Jing , Thurmond Debbie C.
Published: April 2020

Skeletal muscle accounts for ~80% of insulin‐stimulated glucose uptake. The Group I p21–activated kinase 1 (PAK1) is required for the non‐canonical insulin‐stimulated GLUT4 vesicle translocation in skeletal muscle cells. We have recently found that the abundance of PAK1 protein and its downstream effector in muscle, p41‐ARC, are significantly reduced in the skeletal muscle of human type 2 diabetic (T2D) donors. While whole body PAK1 knockout (KO) mice exhibit glucose intolerance and are insulin resistant, it remains unknown as to the relative contribution by the skeletal muscle to this phenotype. As such, we developed an inducible skeletal muscle‐specific PAK1 KO mouse model, as well as an inducible skeletal muscle‐specific PAK1 overexpressor (OE) mouse model, to discern the requirement for PAK1 in skeletal muscle insulin sensitivity, and the molecular mechanisms involved. Indeed, our skeletal muscle‐specific PAK1‐KO and ‐OE mouse studies demonstrate a requirement and role for PAK1 in skeletal muscle insulin sensitivity. Moreover, overexpression of PAK1 in GLUT4‐myc‐L6 myoblasts conferred protection of insulin‐stimulated GLUT4 translocation under insulin resistant conditions. Unexpectedly however, the skeletal muscle‐specific PAK1‐KO and ‐OE mouse models showed impact upon pancreatic function, suggesting that PAK1 expression levels in the skeletal muscle triggered release of an agent that impacted pancreatic responsiveness to blood glucose levels. Testing this concept, conditioned media (CM) collected from PAK1‐overexpressing myotubes was applied to pancreatic β‐cells in culture. Remarkably, β‐cells treated with CM from PAK1‐overexpressing cells showed enhanced glucose‐stimulated insulin release, akin to that seen in the skeletal muscle‐specific PAK1‐OE mice; this enhancement response was selective to PAK1‐derived CM and was not seen with other CM types tested. Taken together, these data suggest that PAK1 levels in the skeletal muscle can exert feedback to the pancreatic β‐cells, unveiling a new molecular mechanism by which PAK1 regulates whole body insulin sensitivity and glucose homeostasis.

Support or Funding Information

This work was supported in part by grants to DCT from the NIH (DK102233, DK067912) and a gift from the Joel Katz fund.
Potential Role of cGAS‐STING Pathway in the Induction of Diabetic Kidney Disease
Authors: Khedr Sherif , Dissanayake Lashodya Vindana , Palygin Oleg , Staruschenko Alexander
Published: April 2020

The role of the cyclic GMP–AMP synthase (cGAS) / Stimulator of interferon genes (STING) / interferon‐β (INFβ) axis in innate immunity has been explored during the last decade. Recently, it has been revealed that the activity of this pathway could be a double‐edged sword; besides protection against invading organisms, its overactivity has been associated with pathological conditions e.g., Aicardi‐Goutieres syndrome (AGS) syndrome. It was also recently reported that activation of the STING‐cGAS pathway had been associated with renal inflammation and fibrosis. However, the impact of the cGAS‐STING pathway on diabetic kidney disease (DKD) has not been studied before. Therefore, the goal of this study was to assess the contribution of the cGAS‐STING‐INFβ pathway towards the development of DKD. We used two established DKD models that develop advanced diabetic kidney lesions ‐ eNOSdb/db mice and type 2 diabetic nephropathy (T2DN) rat. Western blot analysis was used to estimate the expression and/or activity level of different molecules involved in the cGAS‐STING pathway in the kidney tissue from 28 wks old eNOSdb/db mice and corresponding controls. RT‐qPCR was also used to test the transcriptional level of interferon genes, which are the downstream target of this pathway. Comparison between male and female in T2DN rats were used to define changes in the cGAS‐STING signaling pathway during the progression of DKD. As was previously reported, female rats exhibit significant attenuation in progression of diabetes and renal damage in this model. Western blot analysis showed increased expression and/or activity of specific proteins involved in the cGAS‐STING pathway. RT‐qPCR confirmed the Western blot results and showed a significant elevation of the checked INFβ genes. Overall, the activity of cGAS‐STING signaling was higher in males, which is consistent with the protective phenotype of T2DN female rats. In addition, immunohistochemistry (IHC) staining for STING molecule in described rodent models and human renal samples was performed. The results indicate the significant elevation of STING in the renal tissue of both rodents and humans with type 2 diabetes. Therefore, our data revealed that cGAS‐STING pathway is markedly activated in diabetes, and we speculate that inhibiting this pathway would ameliorate the deleterious outcome of the pathologic processes in DKD.
The Effects of Sex Hormone Supplementation on Respiratory Function in Male SCI Rats
Authors: Barok Rebecca J. , Grittner Jessica , Dougherty Brendan
Published: April 2020

Sex hormones significantly influence respiratory function and the expression of respiratory neuroplasticity. For example, 17ß‐ estradiol (E2), the most prevalent and neuroactive sex steroid in females, is necessary for expression of phrenic long‐term facilitation (pLTF), a well characterized form of plasticity induced by acute intermittent hypoxia (AIH) in both females and males. To meet this requirement for E2, males aromatize testosterone to E2 in order to express respiratory neuroplasticity. However, on its own (i.e., without aromatase conversion to E2), testosterone has also been shown to enhance breathing. Since both E2 and testosterone have individually demonstrated the capacity to enhance respiratory function, we hypothesized that supplementation of these sex hormones would improve respiratory function in rats 2 weeks post experimental cervical spinal cord injury (SCI). Adult male Sprague‐Dawely rats received a C2 hemi‐section SCI (or Sham laminectomy) and recovered with standard post‐op care. One‐week post‐injury, a controlled‐release hormone pellet was placed subcutaneously in SCI rats to deliver a pre‐determined dose of daily hormone supplementation for the duration of the study. Pellets contained either E2 (β‐estradiol 3‐benzoate, 10 μg/day), 5α‐dihydrotestosterone (DHT), a testosterone derivative that is not converted to E2 (50 μg/day), or blank placebo pellets (50 μg/day). Respiratory function was measured 7 days later (2 weeks post SCI) using Whole‐Body Barometric Plethysmography. Consistent with prior reports, our preliminary findings demonstrated that placebo treated SCI rats breathed with increased respiratory frequency, but reduced tidal volume production. DHT supplementation had little impact on these respiratory parameters, with values remaining similar to placebo control rats. However, one week of E2 supplementation led to reductions in respiratory frequency and increased tidal volume production during baseline room‐air conditions and in response to a strong respiratory challenge (10.5% O2/5% CO2), suggesting improved overall ventilation. These data suggest that estrogen supplementation may facilitate improvements in breathing function in the early stages of spinal cord injury recovery.

Support or Funding Information

This work was supported by funding from The Minnesota Office of Higher Education, The University of Minnesota Division of Physical Therapy, and The University of Minnesota Department of Rehabilitation Medicine
A Study of the Sinuous Crouch of Smilodon fatalis
Authors: Haji-Sheikh Misty , Naples Virginia
Published: April 2020

Mammalian carnivores generally are considered to belong either to the pursuit or ambush predator categories. Anatomical features for these opposing styles of capturing prey differ, although specific strategies typify familial taxonomic levels. The species of the Canidae tend toward pursuit, while most of the Felidae specialize in ambush. Cats also engage in prey stalking behavior, whether an ambush or pursuit predator. Remaining cryptic for as long as possible is a good strategy to increase the success of a hunt, especially when the quarry sought is fleet of foot, agile, wary and highly capable of evading predation. The extinct sabertooth cat, Smilodon fatalis, has long been considered to be an ambush predator because it was large‐bodied and short‐limbed. Smilodon’s skeleton is robust, and has been reconstructed as heavily muscled because it shows prominent muscle scars. Because of this high degree of robustness, it has also been thought to have had questionable flexibility. No characters showing specific enhancements to Smilodon’s adaptations for clandestine stalking despite its formidable musculoskeletal anatomy have been identified in a search of the published literature. This study describes morphological features that have been newly identified as being able to improve this sabercat’s ability to move sinuously and stealthily during an ambush. These are derived from unique pectoral girdle and forelimb, vertebral column and pelvic girdle and hindlimb features. Specifically, truncated scapular and ilial shapes as well as short limbs give Smilodon a lower crouching profile than can be achieved by other felids. Usually, in sinuously moving cats, even when the animal is seeking to remain as cryptic as possible, the posterodorsal aspect of the scapula and the anterodorsal ilial regions project above the pointed dorsal tips of the elongated vertebral spinous processes. These highest projections may be visible above any vegetation cover, revealing the cat’s presence to potential prey. In contrast, in Smilodon, with fore and hindlimbs equivalently flexed, the shoulder and hip regions fall below the level of the most dorsal tips of the spinous processes, enhancing the cat’s ability to flatten its body to facilitate concealment while stalking. This study interprets the unique morphological limb features in Smilodon as adaptations to enhance the sinuous movements the cat used in getting as close to potential prey animals as possible prior to making a final rush to grapple with them. We also demonstrate how this anatomy leads to the conclusion that Smilodon’s habits fit it to its forested or scrub environment as a solitary ambush predator.
Berberine attenuates cholestatic liver and bile duct injury in Mdr2−/− mice by maintaining bile acid homeostasis
Authors: Wang Yanyan , Xiang Dong , Chen Weidong , Zhao Derrick , Gurley Emily , Wang Xuan , Pu Ge , Tai Yunling , Zhang Yuan , Chen Zhenxia , Wu Jilin , Yan Junkai , Hylemon Phillip B. , Zhou Huiping
Published: April 2020

Background

Cholestatic liver injury is an important clinical problem with a limited understanding of disease pathologies. Modulation of the enterohepatic circulation of bile acids (BAs) may protect against BA‐mediated cholestatic liver injury. Berberine (BBR), a bioactive isoquinoline alkaloid found in a variety of herbs, has been used to treat many diseases, including liver diseases, over hundreds of years. Our previous studies have shown that BBR inhibits high fat‐diet‐induced steatosis and inflammation in rodent models of nonalcoholic fatty liver diseases. However, it remains unknown if BBR has any effect on cholestatic liver injury. The aim of this study is to examine the therapeutic effects and potential mechanisms of berberine on cholestatic liver disease in Mdr2−/− mice, the best‐characterized mouse model of primary sclerosing cholangitis (PSC).

Methods

Mdr2−/− mice with FVB background (100 days old, both male and female) were randomly divided into the control group and BBR group. Mice were treated with BBR (50 mg/kg) or vehicle (0.5% carmellose sodium solution) by intragastric administration once‐daily for eight weeks. Mice were kept in a temperature‐controlled room with 12/12‐hour light/dark cycles and fed ad libitum normal chow diet. At the end of the treatment, the mice were sacrificed. Blood was collected for measuring serum lipids and liver functional enzymes using the Alfa Wassermann Vet ACE Axcel® System. Liver tissues were processed for H&E and Masson’s trichrome staining, immunohistochemistry (IHC), and isolation of total RNA. The mRNA levels of the key genes involved in bile duct proliferation, inflammation, fibrotic injury, and bile acid metabolism were measured by real‐time RT PCR.

Results

BBR treatment significantly improved cholestatic liver injury as indicated by significant reduction of serum levels of ALT, AST, ALP, and BAs, as well as liver to body weight ratios. H&E and Masson's trichrome staining showed a significant reduction of liver fibrosis by BBR treatment. IHC staining of CK‐19 further showered that BBR treatment markedly reduced biliary proliferation and ductular reaction. In addition, BBR treatment reduced hepatic macrophage infiltration. Real‐time PCR results further showed that BBR treatment significantly reduced the mRNA levels of MCP‐1, IL‐1β, α‐SMA, CK‐19 and increased mRNA levels of FXR and SHP in the liver.

Conclusion

BBR significantly reduced cholestatic liver injury and has the potential to be used for the treatment of PSC and other cholestatic liver diseases.

Support or Funding Information

This work was supported by VA Merit Award I01BX004033 and 1I01BX001390, VA Career Scientist Award IK6BX004477 and National Institutes of Health Grant R01 DK104893 and R01DK‐057543
Immunological mechanisms of exosome mediated therapeutic bioactivity in Duchenne muscular dystrophy
Authors: Rogers Russell George , Akhmerov Akbarshakh , Liu Weixin , Sanchez Lizbeth , Marban Eduardo
Published: April 2020

Duchenne muscular dystrophy (DMD) is a lethal myodegenerative disease associated with chronic inflammation. Disruption in the balance between pro‐inflammatory and pro‐regenerative macrophage immunophenotypes contributes to disease progression. Standard‐of‐care is currently limited to anti‐inflammatory steroids, which marginally delay disease progression. We have recently characterized the therapeutic benefits of cardiosphere‐derived cells (CDCs), which are cardiac progenitor/stromal cells with immunomodulatory, pro‐myogenic, and anti‐fibrotic properties. CDCs partially reverse the key abnormalities of heart and skeletal muscle in mdx mice, findings which motivated two clinical trials (HOPE‐Duchenne and HOPE‐2) of CDCs in DMD patients. The bioactivity of CDCs is mediated by the secretion of lipid bilayer nanovesicles called exosomes, which transfer genetic material between cells to modify recipient cell behavior. We previously discovered macrophages are a primary feature in skeletal muscle of CDC‐exosome treated mdx mice, suggesting their presence is not pathological. Here, we sought to elucidate the role macrophages play in the repair of dystrophic skeletal muscle. Circulating monocytes, at least in part, traffic fluorescently‐labeled CDC‐exosomes to damaged muscle after intravenous delivery. Labeled CDC‐exosomes are detected in the interstitium adjacent to non‐muscle cells, macrophages, and surviving muscle fibers. Moreover, CDC‐exosomes confer a unique immunophenotype to mdx macrophages (Mexo). Next generation sequencing and pathway/network analysis indicate Mexo share features of both M1 (pro‐inflammatory) and M2 (pro‐regenerative) macrophages. Indeed, media conditioned by Mexo supports both myoblast proliferation and differentiation in vitro. Antibody chip array indicated Mexo secrete many chemokines and cytokines known to promote inflammatory cell recruitment and modulation. In contrast to M1 and M2 macrophages, Mexo secrete tissue inhibitor of matrix metalloproteinase‐2 (TIMP‐2). Interestingly, TIMP‐2 has been shown to promote myogenic differentiation of C2C12 myoblasts and may function in both ECM remodeling and myogenesis in vivo. Sequencing of small RNAs revealed that several miRNAs with known roles in myogenesis are uniquely enriched in Mexo exosomes. Together, these data suggest CDC‐exosomes are trafficked to areas of muscle damage, in part, by circulating monocytes and modulate the mdx macrophage phenotype to support tissue regeneration. We propose that Mexo macrophages mediate their pleiotropic effects via the secretion of cytokines, chemokines, and possibly exosomes. Modulation of mdx macrophage functions by CDC‐exosomes, to support muscle health, may represent a novel approach to treating DMD.

Support or Funding Information

Muscular Dystrophy Association, Coalition Duchenne, NIH R01 to EM