bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of Dihydroxylated Fatty Acid Mediators on Ethanol‐Induced Liver Injury in a Mouse Model of Alcoholic Liver Disease
Authors: Warner Jeffrey Barr , Hardesty Josiah , Warner Dennis , Song Ying , McClain Craig , Kirpich Irina 
Published: April 2020

BackgroundAlcoholic liver disease (ALD) is the most common chronic liver disease and a major contributor to liver disease burden worldwide, and a disease for which there is no FDA‐approved treatment. The mechanisms of ALD pathogenesis are not completely understood. Data from our group indicate that dihydroxylated fatty acids (dihydroxy‐FAs) are increased in the livers of human alcoholic hepatitis patients. Dihydroxy‐FA lipid mediators are produced via cytochrome P450/epoxide hydrolase‐mediated metabolism of polyunsaturated fatty acids and are generally pro‐inflammatory. Despite their increase in human ALD, the role of dihydroxy‐FAs in ALD has not been explored.HypothesisExogenous administration of dihydroxy‐FAs will exacerbate liver injury in a mouse model of ALD.MethodsMale C57BL/6 mice were provided ethanol (EtOH) using a chronic‐binge Lieber DeCarli ad libitum EtOH feeding model. Mice were administered either 8,9‐DiHETE (arachidonic acid metabolite), a combination of 9,10‐DiHOME and 12,13‐DiHOME (linoleic acid metabolites), or a vehicle control by i.p. injection once daily for two days prior to sacrifice. Liver injury, inflammation, and steatosis were measured. To further elucidate the effects of dihydroxy‐FAs on liver cells, primary mouse hepatocytes or HepG2 cells were treated with 8,9‐DiHETE, 9,10‐DiHOME, 12,13‐DiHOME, or a vehicle control. Effects on baseline or palmitic acid (PA)‐induced cell death were measured by MTT assay and apoptotic cell death was identified by Annexin‐V staining (cellomics).ResultsIn vivo, 8,9‐DiHETE treatment modestly while 9,10/12,13‐DiHOME significantly increased liver injury as determined by elevated plasma ALT levels (1.3‐fold and 2.2‐fold p=0.04, respectively). While 8,9‐DiHETE had no effect on steatosis, 9,10/12,13‐DiHOME decreased liver triglyceride/free fatty acid levels compared to vehicle control. Dihydroxy‐FA treatment decreased the hepatic expression of several pro‐inflammatory chemokines at the mRNA level, including Mip2a and Mcp1. In vitro, 8,9‐DiHETE treatment of HepG2 cells or primary mouse hepatocytes significantly exacerbated PA‐induced cell death as determined by MTT assay, whereas 9,10‐DiHOME, and 12,13‐DiHOME had no effect on PA‐induced or baseline cell death or apoptosis.ConclusionsDifferent dihydroxy‐FAs have diverse effects in vivo, in a mouse model of ALD, and in vitro. In vivo, dihydroxy‐FAs exacerbated liver injury and was associated with down‐regulated several pro‐inflammatory cytokines. In vitro, 8,9‐DiHETE increased hepatocyte cell death, while 9,10/12,13‐DiHOME showed no effects. Future studies are warranted to dissect the effects of individual dihydroxy‐FAs in ALD.Support or Funding InformationThis work was funded by grants from the National Institutes of Health and the Department of Veterans Affairs.
PaeR, a Multiple Antibiotic Resistance Regulator Homolog from Clostridium difficile is a Sequence‐Specific DNA Binding Protein that Responds to Small Molecule Effectors
Authors: Thompson Kara , Garcia-Ortiz Ruben , Wilkinson Steven 
Published: April 2020

Proteins belonging to the multiple antibiotic resistance regulator (MarR) family are ubiquitous amongst species in the bacterial and archaeal domains. These proteins function as transcriptional regulators of a wide variety of cellular processes including catabolic pathways, oxidative stress response mechanisms, efflux or degradation of toxins, and virulence factor expression. DNA binding by MarR proteins is mediated by a characteristic winged helix‐turn‐helix (wHTH) motif which makes sequence‐specific contacts with both the DNA major and minor grooves. For a number of MarR homologs, DNA binding is abrogated by ligand binding. The natural ligands of MarR proteins are structurally diverse but generally fall into three main categories: small aromatic compounds, metals and reactive oxygen species. In this study, we report the first biochemical investigation of a MarR homolog from the pathogenic bacterium Clostridium difficile which we have named PaeR (putative antibiotic efflux regulator). Biophysical investigations of the structural properties of PaeR indicate that this protein exists predominantly as a homodimer in solution and unfolds in a manner consistent with a simple, two‐state transition. PaeR binds at two sites in its own promoter/operator region, suggesting autoregulation. Analysis of binding at a single site indicates a high‐affinity interaction with a microscopic dissociation constant of 3.3 ±0.4 nM. Competitive electrophoretic mobility shift assays (EMASs) indicate that PaeR interacts with DNA in a highly sequence‐specific manner. Ligand binding studies indicate that PaeR binding to its cognate DNA binding sequence is attenuated by small aromatic compounds. These findings, in conjunction with the genetic context of paeR in the C. difficile genome, suggests a possible role of this MarR homolog in regulating antibiotic resistance in this pathogen.Support or Funding InformationBill and Linda Frost Fund
Characterization of the Dual Routes for Bacillus subtilis Aspagainyl‐tRNA Synthesis
Authors: Cassello Natalie , Sheppard Kelly 
Published: April 2020

Attaching asparagine to its cognate transfer RNA (tRNAAsn) is an essential step in protein synthesis. The bacterium Bacillus subtilis encodes two distinct routes for the formation of asparagingyl‐tRNAAsn. In the first pathway, asparaginyl‐tRNA synthetase catalyzes the direct esterification of asparagine tRNAAsn. In the second pathway, a non‐discriminating aspartyl‐tRNA synthetase misaminoacylates tRNAAsnwith aspartate. The aspartyl‐tRNAAsnformed is then transamidated by the amidotransferase GatCAB to form asparaginyl‐tRNAAsn. The non‐discriminating aspartyl‐tRNA synthetase, GatCAB, and tRNAAsnform a complex known as the transamidosome to synthesize Asn on tRNAAsn. Intriguingly, Bacillussubtilis also codes for two distinct tRNAAsnisoacceptors in its genome. Why Bacillus subtilis acquired an asparaginyl‐tRNA synthetase is not clear. We hypothesize that it enables the organism to adapt to different environmental conditions. To test this hypothesis, we have purified the Bacillus subtilis asparaginyl‐tRNA synthetase and transamidosome. We will report on characterizing in vitro the two pathways for asparaginyl‐tRNA synthesis under different physiologically relevant conditions as well as knocking out asnS in the Bacillus subtilis genome that encodes asparaginyl‐tRNA synthetase. The work will provide insight into how bacteria have adapted amino acid metabolism and use in protein synthesis for different environmental niches.Support or Funding InformationNational Science Foundation (MCB‐1615770)
Investigating the Activity of Novel Sigma‐2 Receptor Analogs in Pancreatic Ductal Adenocarcinoma Cell Lines
Authors: Inbody Laura R. , Hollenbacher Courtney A. , Hickey Mikaela C. , Kinder David H. , Schneider Ryan A. 
Published: April 2020

Pancreatic cancer is an aggressive cancer that is highly resistant to most chemotherapy agents and has an estimated 5‐year survival rate of less than 10%. Sigma‐2 receptors (S2Rs) are highly upregulated in many cancers, including pancreatic. Specifically, S2Rs are overexpressed in pancreatic cancer cells, while minimally expressed in normal pancreatic cells, which makes them a potentially selective drug target. Additionally, pancreatic ductal adenocarcinoma cell (PDAC) lines have been found to be sensitive to SR2‐ligand binding, which initiates an apoptotic response. The selective estrogen receptor modifier, tamoxifen, has been shown to have affinity for S2Rs. We developed a series of analogs based on tamoxifen’s scaffold, with the intent to increase sigma‐2 receptor activity while decreasing estrogen receptor affinity and activity.Previously, we have shown the analogs in our sigma receptor library presented here have low micromolar IC50 values in glioblastoma, melanoma, breast, and colorectal cancer cell lines. These analogs do not display selectivity between cancerous and noncancerous cell lines of normal human astrocytes and mammary epithelium.Here we present additional screening data of SP‐1, SP‐5, SP‐6, and SP‐9 in three PDAC lines, ASPC‐1, BXPC‐3, and Su86.86. SP‐1, SP‐5, and SP‐9 all demonstrate similar efficacy with IC50 values ranging from 30 to 50 μM in each of the three PDAC lines. These findings are in line with previous studies with this library in the other cell lines listed above. Interestingly, the SP‐6 analog was much less active with IC50 values of 100 μM or greater in all three PDAC lines.In summary, we found that our sigma‐2 analogs have low micromolar IC50 values in multiple PDAC lines. In the future, we would like to assess selectivity against non‐cancerous pancreatic cells and measure binding affinity of these analogs for S2R and estrogen receptors. Lastly, we would like to explore the mechanisms by which these analogs induce cell death in pancreatic cells.
The Role of Autophagy in Hydrogen Peroxide‐ or Methylglyoxal‐induced Cellular Damage in H9c2 Cells
Authors: Sehgal Amogh , Onal Halil , Martorana Rose , Wastella Catherine , Pjetergjoka Adona , Barsotti Robert , Young Lindon , Chen Qian 
Published: April 2020

Oxidative stress (e.g., increased hydrogen peroxide [H2O2] levels) and dicarbonyl stress (e.g. increased methylglyoxal [MG]) serves as an initiator for the pathogenesis of cardiovascular diseases and diabetic complications. Increased H2O2‐derived free radicals and MG can be highly reactive and attack cellular components resulting in cell damage and even cell death. Normally, endogenous antioxidant mechanisms and glyoxalase degrade H2O2 and MG, respectively. Additionally, autophagy facilitates the break‐down of damaged cellular components allowing the components to be recycled by the cells. Researchers are still unclear about whether autophagy exacerbates cell damage or protects cells under higher doses of H2O2 and MG. This study investigated cell damage and autophagy changes when rat cardiac H9c2 myoblasts were treated with H2O2 or MG. Moreover, autophagy enhancing or inhibiting drugs were tested to evaluate their effects on H2O2‐ or MG‐induced cell damage. We found that H2O2 (300–900 mM, n=4) dose‐dependently decreased cell viability via measuring live cell dehydrogenase activity. H2O2 (600 mM, n=4) decreased cell viability to 16 ± 1% of the control. Similarly, MG (400–1400 mM, n=5) decreased cell viability in a dose‐dependent manner. MG (1200 mM, n=5) decreased cell viability to 29 ± 6% of the control. These results were further confirmed with the use of double staining, which contained calcein‐AM for labeling live cells and propodium iodide for labeling dead cells. By using Cyto ID autophagy detection dye, we found that H9c2 cells exhibited higher autophagy fluorescence after incubation of H2O2 (300–600 mM, n=2) or MG (400–1200 mM, n=2). Furthermore, trehalose (0.1–100 mM, n=3), an autophagy enhancer, reduced MG (1200 mM) induced cell damage. Trehalose (100 mM, n=3) increased cell viability by 69 ± 27% when compared to MG (1200 mM). Our preliminary results suggest that higher doses of H2O2 and MG can cause cell damage accompanied with increased autophagy. The autophagy enhancer trehalose provided cell protection against MG. Further studies are needed to confirm the effects of autophagy modulation on H2O2 and MG‐induced cell damage by using different autophagy modulators.Support or Funding InformationThis research was supported by the Division of Research, Department of Biomedical Sciences, and the Center for Chronic Disorders of Aging at Philadelphia College of Osteopathic Medicine.
Role of focal adhesion kinase in burn induced gut epithelial barrier dysfunction
Authors: Wang Fang , Eitnier Rebecca A. , Aponte Alexandra M. , Ewing Thomas J. , Wu Mack H. 
Published: April 2020

Mucosal barrier failure is a critical pathological step in the development of multiple organ failure during severe burn injury and trauma. The opening of the epithelial barrier leads to increased leukocyte infiltration into the lamina propria and microbial translocation from the intestinal lumen. These events augment intestinal inflammation and contribute to the severity of trauma‐associated complications. The aim of the study was to characterize the role of focal adhesion kinase (FAK) in intestinal barrier dysfunction following thermal injury. Our data demonstrated that severe thermal injury significantly increased the gut lumen‐to‐blood transport of FITC‐dextran in mice. The effect was significantly attenuated by the inhibition of FAK with PF573228. Stimulation of mouse intestinal epithelial cells with TNFα/IFNγ caused a decrease in transcellular electric resistance coupled with downregulation of junctional proteins. The responses were prevented by the inhibition of either PTK6 or FAK. Western blot analysis showed that knocking down PTK6 in cells reduced TNFα/IFNγ mediated FAK activation via diminished detection of FAK phosphorylation at Y397. Reintroduction of active PTK6 to PTK6 deficient cells rescued FAK phosphorylation in response to TNFα/IFNγ. The results suggest that PTK6 dependent FAK phosphorylation plays an important role in gut barrier dysfunction during inflammatory injury.Support or Funding InformationVA BX000799, NIH HL120954
Genetic Background influences Endothelial Function along the Mouse Vascular Tree
Authors: Holly Dylan Stone , Massett Michael , Gaytan Samantha , Kim Hyoseon , Shin Songyi , Woodman Christopher 
Published: April 2020

The endothelium is crucial for its role in maintaining vascular integrity and protecting against cardiovascular disease. Endothelial dysfunction is associated with a host of disease states including cardiovascular disease, diabetes, chronic kidney disease, and atherosclerosis. However, it is unclear whether endothelial function or dysfunction varies throughout the vasculature. In addition, accumulating evidence suggests that endothelial function, and more generally vasomotor function, is genetically regulated. The purpose of this study was to determine the influence of genetic background on vasomotor function in blood vessels of differing size. Vasomotor performance was measured in aorta (thoracic (TA), and abdominal (AA)) and conduit arteries (carotid artery (CA) and femoral artery (FA)) from four inbred strains (SJL/J (SJL), DBA/2J (DBA), NZW/LacJ (NZW) and C57BL/6J (B6)) of mice. Arteries were dissected, cut into 2 mm segments, and mounted in a wire myograph system. Increasing concentrations of phenylephrine (PE, 1×10−9 – 1×10−5M) were used to measure contractile responses, while increasing concentrations of the endothelium‐dependent vasodilator acetylcholine (ACh, 1×10−9 – 1×10−5M) and endothelium‐independent vasodilator sodium nitroprusside (SNP, 1×10−9 – 1×10−5M) were used to assess relaxation responses. Two way ANOVA revealed a significant main effect of strain in response to PE for AA (P = .0032) and CA (P = .0098). Post hoc analysis for max contractile response showed impaired vasoconstriction in NZW AA compared to B6 (P = .0004). In response to ACh, a significant main effect of strain was found for all vessels (TA: P = <.0001, AA: P = .0156, CA: P = .0069, FA: P = <.0001). Post hoc analysis showed that TA max vasorelaxation was impaired for all strains (NZW: P = < .0001, DBA: P = .0140, SJL: P = < .0001) and that NZW FA were impaired when compared to B6 ( P = .0015). A significant main effect of strain in response to SNP was found in TA (P = .0011), AA (P = .0086), and CA (P = < .0001). Relaxation responses to SNP were not significantly different between strains. These findings indicate a strong influence of genetic background on endothelial function and could be important in understanding the implications that genetics have on vascular response to vasoactive agents.Support or Funding InformationThis work was supported by a Texas A&M Triads for Transformation grant (MPM), a Texas A&M Merit Fellowship (DH), a Texas A&M College of Education Strategic Research Award (SYS), and J.L. Huffines Institute of Sports Medicine and Human Performance (SYS).
SPARC: Development of Pigs with mCherry‐CRE Recombinase Fusion Proteins in Cholinergic Neurons
Authors: Sponchiado Mariana , Liao Yan-Shin , Collins Emily , Whitworth Kristin M. , Wells Kevin G. , Prather Randall S. , Reznikov Leah R. 
Published: April 2020

Large animals are increasingly utilized in biomedical research and offer an advantage in that their anatomy and physiology closely parallel humans. Yet despite these advantages, rodent models dominate many areas of research, including neuroscience. This might be due in part to the greater number of technologies available to rodent researchers. Here we describe the development of pigs with mCherry and CRE‐recombinase proteins expressed in cholinergic neurons. We first cloned a 5,972 bp DNA fragment containing the predicted choline acetyltransferase (ChAT) promoter from porcine genomic DNA and placed into a pGlow TOPO™ TA expression system. We then tested for promoter activity by transfecting the expression system into porcine hippocampal slices and assessed for green fluorescent protein (GFP). We observed GFP at 60–72 hours post transfection. Confirmation of specificity was achieved by using antibody detection of ChAT. To optimize the expression system, we relied upon proven strategies used in rodents, and added an additional 535 bp of predicted promoter to the 5’ end. We then used this sequence to drive production of a mCherry‐P2A‐CRE recombinase fusion protein (ChAT‐mCherry‐P2A‐CRE). We transfected brain slice cultures from the pig hippocampus, striatum and brainstem with ChAT‐mCherry‐P2A‐CRE. Approximately 60 hours later, we isolated RNA and froze brain slices from transfected and non‐transfected samples. qRT‐PCR revealed mCherry and CRE recombinase mRNA in all transfected tissues. Expression of mCherry protein was also visible in discreet subsets of neurons in transfected samples. Studies are ongoing to confirm mCherry expression in cholinergic neurons. Key next steps include engineering our ChAT‐mCherry‐P2A‐CRE to contain GGTA1 homology arms for placement into the GGTA1 safe harbor in the pig genome, providing in vivo validation of ChAT‐mCherry‐P2A‐CRE using lentivirus pseudotyped with rabies glycoprotein, and targeting porcine fetal fibroblasts for somatic cell nuclear transfer. If successful, this model will allow for simultaneous visualization of neurons (and their innervation), and the ability to control or monitor neural activity through CRE‐mediated recombination.Support or Funding InformationHL119560, 1OT2OD026582
Intraspecific and intrafamilial variation in primate forearm muscle architecture
Authors: Hartstone-Rose Adam , Boettcher Marissa L. , Seelye Jason , Worden Nikole , Dickinson Edwin 
Published: April 2020

Recent studies have examined locomotion and postural correlates of primate forearm muscle architecture. However, these have done so using relatively small sample sizes – usually one or two specimens per species, and relatively few species per genus and family. Thus, little is known about intraspecific variation in forearm muscle fiber architecture in primates, and whether there are notable differences in these variables between closely related taxa. The current study addresses these deficits by assessing both qualitative (presence/absence of specific muscles) and quantitative (physiological cross‐sectional area [PCSA], fascicle length [FL] and muscle mass [MM]) differences in forearm muscle architecture of larger intraspecific and samples with greater coverage of specific primate families than had been included in previous work. Namely, we studied 30 callitrichid specimens from 11 species, 15 lemurid specimens from 7 species and 9 cebids from two species ‐ a total sample size of 54 specimens from 20 species. Within species, FL varied the greatest among architectural variables and MM varied the least. We also found that there is great variation in both the extent of separation and fusion of flexor digitorum superficialis and profundus, and in the extent of accessory extensors in most species of callitrichids and Saimiri, but no variation in the gross forearm muscle architecture within lemurids. More than a quarter of the callitrichid specimens had forearm muscle configurations that deviate from their standard published description, suggesting that blanket characterizations of anatomical configurations at the genus or family level may obscure potentially important intra‐ and inter‐specific trends.Support or Funding InformationNational Science Foundation: IOS‐15‐57125, BCS‐14‐40599
Autophosphorylation of the CK1 kinase domain regulates enzyme activity and function
Authors: Cullati Sierra , Chen Jun-Song , Gould Kathy 
Published: April 2020

CK1 enzymes are conserved, acidophilic serine/threonine kinases with a variety of critical cellular functions; misregulation of CK1 contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Despite this, little is known about how CK1 activity is controlled.CK1 kinases have highly similar catalytic domains, plus a conserved extension to that kinase domain that is important for enzyme stability and activity. In contrast to the catalytic domains, the C‐terminal tails of CK1 family members diverge in sequence and length; however, they all appear to serve as substrates of autophosphorylation. The autophosphorylated tails are proposed to inhibit kinase activity by acting as pseudosubstrates. In addition to C‐terminal autophosphorylation, autophosphorylation of the CK1□ kinase domain has been detected, but its effect on CK1 activity and cellular function has never been explored.Here, we addressed the role of kinase domain autophosphorylation in human CK1□ and CK1□, as well as Hhp1 and Hhp2, their homologues in Schizosaccharomyces pombe. In each case, we found that autophosphorylation of a conserved threonine residue in the kinase domain inhibited enzyme activity. This site resides in the mobile L‐EF loop proximal to the active site, distinct from the well‐characterized T‐loop autophosphorylation that occurs in other kinase families. We found that yeast and human enzymes with phosphoablating mutations at this site are hyperactive in vitro. In vivo, mutation of this site protects yeast cells from heat shock, indicating a change in substrate profile, a prediction we are currently testing using quantitative phosphoproteomics. We have also found that autophosphorylation of the kinase domain may affect autophosphorylation of the C‐terminus, so there is likely interplay between the two different modalities of CK1 autoregulation that ultimately determines the extent to which substrates are phosphorylated.We propose that phosphorylation on the L‐EF loop prevents substrate docking with the kinase domain by shielding the positively charged substrate binding pocket and/or sterically hindering the active site. Due to the strong sequence conservation of this autophosphorylation site and the functional importance of the L‐EF loop, which is unique to the CK1 family of kinases, this mechanism is likely to regulate the majority of CK1 enzymes in vivo.Support or Funding InformationThis work was supported by R35‐GM131799 (to KLG) and T32‐CA119925 (to SNC).