bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of L382 and S383 in the βDELSEED‐motif of ATP synthase
Authors: Steiner Amanda Jayne , Ahmad Zulfiqar 
Published: April 2020

BackgroundAntibiotic 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.MethodGrowth 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.ResultsOur 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.ConclusionsWe 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 InformationThis 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 InformationUWL 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 InformationR01HD067541‐06, P20GM121334, AHA 19CDA34670055, T32HL105324
Sex‐Differences in Gastric Smooth Muscle Function: Role of G Protein‐Coupled Estrogen Receptor
Authors: Mahavadi Sunila 
Published: April 2020

The regulation of gastrointestinal (GI) motility by estrogen and its receptors has been a topic of great interest owing to sexual dimorphism that exists in motility disorders. Scientific and clinical evidence establishes the fact of GI motility disturbances during estrous cycle but, there is a gap in the knowledge and understanding of estrogen and its receptors role in GI motility physiology during these cycles. Estrogen activates classical nuclear receptors ERα, ERβ and atypical 7‐transmembrane G protein‐coupled estrogen receptor (GPER or GPR30) to initiate both genomic and non‐genomic effects. GPER is involved in the several rapid cellular signaling events. The pleiotropic effects manifested by GPER action include regulation of body weight, glucose homeostasis, anti‐inflammation, anti‐nociception, and vascular muscle relaxation. Ablation of GPER leads to increase in mean arterial pressure in female mice, whereas treatment with GPER‐specific agonist G‐1 lowers arterial blood pressure in hypertensive and ovariectomized female rats. The role of GPER in mediating estrogen effects on GI smooth muscle is unknown.AimTo understand the role of GPER in gastric smooth muscle function in male and female mice.MethodsMuscle strips and muscle cells were isolated from the stomachs of control and GPER knockout mice. Expression of GPER was analyzed by qRT‐PCR and western blot. cAMP levels in response to estrogen and G‐1 was measured by radioimmunoassay. Relaxation was measured in organ bath studies in muscle strips and by scanning micrometry in isolated muscle cells.ResultsExpression of GPER (mRNA and protein) in the gastric smooth muscle was higher in female mice compared to male mice. 17β‐estradiol and G‐1 caused an increase in cAMP levels and inhibited acetylcholine‐induced muscle contraction (i.e., caused muscle relaxation) in gastric muscle strips and isolated muscle cells from both female and male mice, however, the increase in cAMP levels and the amount of relaxation was significantly greater in gastric muscle from female mice compared to male mice. The effect of 17β‐estradiol and G‐1 on muscle relaxation was concentration‐dependent. Relaxation was blocked by the GPER specific antagonist G15 (10 μM) in gastric muscle from control mice and absent in gastric muscle from GPER knockout mice. Relaxation was higher in proestrus and estrus phases compared to diestrus phase in female mice and this is consistent with higher expression of GPER in proestrus and estrus phases compared to diestrus phase.ConclusionActivation of GPER causes relaxation via cAMP/PKA pathway in gastric smooth muscle from female and male mice. Greater relaxation in gastric smooth muscle from female mice is due to higher expression of GPER in female mice compared to male mice. Pilot studies suggests that higher expression of GPER in the stomach of female mice is due to differential regulation of GPER expression by epigenetic mechanisms.Support or Funding InformationSupported by grants from VETAR (SM); IACUC approval AD10001201
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 InformationThis work was supported in part by grants to DCT from the NIH (DK102233, DK067912) and a gift from the Joel Katz fund.
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 InformationThis 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
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

BackgroundCholestatic 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).MethodsMdr2−/− 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.ResultsBBR 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.ConclusionBBR 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 InformationThis 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 InformationMuscular Dystrophy Association, Coalition Duchenne, NIH R01 to EM