bluuebunny/crossref_metadata_2025_split huggingface dataset:
Direct Observation of APT Protein Dynamics Using Tryptophan Fluorescence
Authors: Zeheralis Chris Louis , Johnson R. Jeremy 
Published: April 2020

Human acyl‐protein thioesterases (APTs) catalyze the depalmitoylation of key signaling proteins attached to the plasma membrane. Catalysis was hypothesized to be regulated by the large‐scale movement of an essential dynamic loop, which transitioned the protein between active and inactive states. However, the causes for the movement of this loop are unknown. Herein, we investigated several potential factors that trigger loop movement in APTs. Specifically, we measured the effect of two irreversible serine hydrolase inhibitors and various membrane lipid models on this dynamic loop movement using intrinsic tryptophan fluorescence. Using a library of combinatorial tryptophan variants of human APT1 and APT2, we followed dynamic changes in their structure in response to shifting ligand and membrane incubations. Amongst these various conditions, the APT specific inhibitor Palmostatin B showed the largest specific effect on loop dynamics. Relating these loop dynamics to sites of modification allowed us to identify distinct regions of APTs interacting with inhibitors and with membranes.Support or Funding InformationFunded by NSF MCB‐1812971
The Roles of Inducible Nitric Oxide Synthase in Mediating the Effects of Embryotoxic Cytokines on Mouse Embryonic Cells
Authors: Guo Yanlin , Chen Bohan , Fendereski Mona , Hargis McKenzie 
Published: April 2020

An early embryo is the most vulnerable stage in the life cycle of mammals, and it faces various immunologic challenges during the course of pregnancy. In particular, the blastocyst (the pre‐implantation embryo) is exposed to inflammatory cytokines resulting from the implantation process. Embryonic cells in the blastocyst have a dedicated task of rapid cell proliferation; how they deal with inflammatory cytokines known to negatively affect cell viability and proliferation is an important yet poorly understood question. We have investigated the effects of TNFα and IFNγ, two “embryotoxic cytokines” that impair blastocyst development and the process of implantation, on embryonic cells. Our results suggest that embryonic stem cells (ESCs), the pluripotent stem cells derived from the inner cell mass of the blastocyst, are insensitive to the cytotoxicity of TNFα and IFNγ. However, when ESCs are differentiated to fibroblasts (ESC‐FBs), they become susceptible to the cytotoxicity of TNFα and IFNγ. In particular, the combination of TNFα and IFNγ (TNFα/IFNγ) significantly reduced cell proliferation and induced apoptosis of ESC‐FBs. The cytotoxic effect of TNFα/IFNγ parallels the induction of nitric oxide synthase (iNOS) in ESC‐FBs, which has been known to cause cellular damage by producing nitric oxide (NO) as a free radical. In contrast, TNFα/IFNγ does not induce the expression of iNOS in ESCs or exert any detectable negative effects on cell proliferation and pluripotency of ESCs. However, ESCs are sensitive to the cytotoxicity of sodium nitroprusside, which can chemically generate NO in cell culture. Together, our results suggest that iNOS is a key effector molecule that mediates the cytotoxicity of TNFα/IFNγ in ESC‐FBs, but not in ESCs. Therefore, the lack of iNOS induction by TNFα/IFNγ in ESCs may be a protective mechanism that allows ESCs to avoid the cytotoxicity of the two cytokines. We are currently investigating the underlying molecular mechanisms and the biological implications of this finding.Support or Funding InformationThis work was in part supported by the National Institute of General Medical Sciences (R15GM128196‐01). We thank Mississippi‐IDeA Network of Biomedical Research Excellence for the use of the imaging facility (funded by the National Institute of General Medical Sciences P20 GM103476‐11).
Acute eCig Vapor or SHS Exposure Induces Inflammatory Signaling in the Adult Murine Lung
Authors: Hirschi-Budge K. , Tsai K. Y.F. , McCusker H. Graviet , Homer K. , Rock S. , Davis T. , Llavina S. , Fowers R. , Long M. , Jensen T. , Arroyo J. , Graff T. , Reynolds Paul R. 
Published: April 2020

The detrimental effects of exposure to tobacco smoke have been confirmed repeatedly over the last several decades. Primary smoking produces the greatest damage; however, secondhand smoke (SHS) is also notably detrimental. More recently the development of electronic cigarettes (eCigs) has introduced a new aspect to the study of smoke exposure. A heating element is used by eCigs to vaporize an oil‐based carrier that contains nicotine and various flavorings. eCig liquid and aerosols have been linked to increased cytotoxicity, inflammation, and production of reactive oxygen species. Negative effects on respiratory health have also been confirmed. Both tobacco smoke and eCig vapor have been found to alter cellular signaling and activate inflammatory responses. Of note, inflammatory pathways include signaling through receptors for advanced glycation end‐products (RAGE). Our objective in this study was to examine effects of acute exposure to eCig vapor compared to SHS in the murine lung. Wild type mice were exposed to vapor containing Apple or Cinnamon flavoring, or to SHS for a period of four days using a nose‐only delivery system (Scireq). Exposure consisted of 30 minutes of eCig vapor or SHS generated by three cigarettes over a period of 30 minutes. Control mice were exposed to room air. Animal use was in accordance with approved IACUC protocols at Brigham Young University. After exposure, lung tissue was collected: the left lung lobe was inflated for histological analysis, and the right lung lobes were snap frozen for protein and RNA analysis. H&E stained lung sections did not show changes in morphology and staining for pulmonary cell types using cell‐specific antibodies (SPC, CCSP, T1a and Fox J1) revealed evidence of cell population alterations potentially explaining increases in pro‐apoptotic cleaved caspase‐3 following exposure. Immunoblotting for RAGE and signaling intermediates activated by RAGE such as NF‐kB and pERK revealed differential expression following eCig or SHS exposure. These results indicate that activation of inflammatory signaling pathways occurs following an acute four‐day exposure. Such signaling in eCig vapor exposed mice has potential to lead to chronic inflammatory disease characteristics with longer exposures potentially leading to chronic obstructive disorders resulting from tobacco smoke exposure.Support or Funding InformationThis work was supported by a grant from the Flight Attendant’s Medical Research Institute (FAMRI, PRR and JAA) and a BYU Mentoring Environment Grant (JAA and PRR).
Total and Afferent Renal Denervation Blunts Hypertension and Central and Renal Inflammation in the Developmental Phase of 2‐Kidney, 1‐Clip Hypertension
Authors: Lauar Mariana Ruiz , Van Helden Dusty , Banek Christopher T. , Evans Louise C. , Menani José Vanderlei , Osborn John W. 
Published: April 2020

Hypertension (HTN) is a multifactorial disease that affects one third of the population of western countries. Although 95% of cases are of unknown origin (i.e., primary HTN), it is well established that renal ischemia results in secondary renovascular HTN. Experimental renovascular HTN is induced in rats by placing a silver clip on the left renal artery creating a stenosis, which decreases renal perfusion pressure and increases renin release from the clipped kidney (2K1C‐HTN). Previous studies indicate a role of renal nerves in 2K1C‐HTN since renal denervation (RDN) attenuates HTN in 2K1C‐rats. Moreover, activation of the sympathetic nervous system in this model is associated with inflammation in the hypothalamus, a key site for regulation of sympathetic activity and an area that also receives input from afferent renal nerves. In the present study, we tested the hypothesis that renal nerves modulate the levels of hypothalamic and renal proinflammatory cytokines and mean arterial pressure (MAP) during the development of 2K1C hypertension in rats. Male Sprague‐Dawley rats (150–180 g, n=5–8/group) were instrumented with telemeters to measure MAP. One week later a clip was placed on the left renal artery of the rats to generate 2K1C HTN. In the same surgery rats also underwent either total or afferent RDN (2K1C‐TRDN or 2K1C‐ARDN) of the clipped kidney. In sham rats (2K1C‐sham) the left renal artery was exposed but not denervated. MAP was continuously measured for the following 6 weeks after which rats were euthanized and brains and kidneys removed for measurement of hypothalamic and renal cytokine content. MAP at the end of the study was 160±6 mmHg in 2K1C‐sham rats. In contrast, MAP was significantly lower in both 2K1C‐TRDN (135±7 mmHg) and 2K1C‐ARDN (133±7 mmHg) rats. TRDN and ARDN statistically reduced hypothalamic protein levels of TNF‐α (0.24±0.01 and 0.2±0.01 pg/mg, respectively) compared to 2K1C‐sham rats (0.3±0.02 pg/mg). Similarly, ARDN statistically reduced hypothalamic IL1β, IL‐2 in 2K1C rats. TRDN trended to the same effect but this was not statistically significant. Renal inflammatory cytokines/chemokines (MCP‐1, IL‐6, TNF‐α) were increased in clipped compared with non‐clipped kidney in 2K1C‐rats. Moreover, ARDN and TRDN statistically reduced this inflammatory response. These results suggest that RDN decreases hypothalamic and renal inflammation in 2K1C hypertensive rats and this may be one mechanism by which RDN decreases MAP in the developmental phase of the 2K‐1C hypertension model.Support or Funding InformationNIH R01 HL116476, FAPESP (2018/11221‐1) and PROPE/FUNDUNESP
Evidence of the Intrinsic Coagulation Pathway and Contact System Activation in Sickle Cell Patients During Steady State
Authors: Pawlinski Rafal , Henderson Michael W. , Ilich Anton , Sparkenbaugh Erica , Key Nigel S. 
Published: April 2020

A chronic hypercoagulable state and increased risk for venous thromboembolism are prominent features of sickle cell disease (SCD). This hypercoagulable state is mediated by activation of both the intrinsic and extrinsic coagulation pathways. There is growing interest in targeting components of the intrinsic coagulation pathway (factor XII and XI) to prevent thrombotic disorders, primarily because this goal may be attainable without incurring any significant bleeding risk. In addition, FXIIa can also activate plasma prekallikrein to kallikrein (PK), which initiates the contact pathway. We have previously reported that FXIIa contributes to the prothrombotic state and vascular inflammation in sickle mice. In this study we investigated if the intrinsic and contact pathways are activated in SCD patients.Fifty four African American outpatients with SCD and twenty three healthy African‐Americans were recruited. The study was approved by the University of North Carolina’s Institutional Review Board for human subjects. Platelet‐poor plasma was prepared from blood drawn into 3.2% sodium citrate (9:1). To detect activation of FXII, FXI, FIX and PK we have developed sandwich ELISAs to quantitate plasma levels of complexes formed between active forms of these proteases and their natural inhibitor C1 esterase inhibitor (C1). We used mouse monoclonal antibodies specific to the proteases to capture the complexes and a sheep polyclonal antibody to recognize C1. The polyclonal antibody is biotinylated, and detected with streptavidin‐horseradish peroxidase. Protein standards are generated by combining ~6μM purified proteases with ~12μM recombinant C1 for 18 hours at 37C, and quenched with 25μM PPACK. These standards are diluted to achieve the working linear range of the assay.Compared to healthy controls, plasma levels of FXIIa‐C1 (1.07±0.19 vs 2.33±0.29nM; mean ±SEM), FXIa‐C1 (0.47±0.09 vs 0.98±0.10nM) and FIXa‐C1 (0.84±0.13 vs1.43±0.16nM) were all significantly elevated in SCD patients (p<0.05 for all groups). Similar to that plasma levels of PK‐C1 complexes were also elevated in SCD patients (12.50±1.06 vs 16.74±1.05; p<0.05). In the SCD patient group, highly significant correlations were observed between activation of FIX and activation of both upstream components of the intrinsic pathway (r=0.91; p<0.0001 for FXIIa and r=0.91; p<0.0001 for FXIa). Interestingly, a significant correction was also observed between FIX‐C1 and PK‐C1 complexes (r=0.64; p<0.0001).Our data strongly suggests that both the intrinsic coagulation pathway and contact system are activated and contribute to the chronic hypercoagulable state observed during steady state in SCD patients. The contribution of these two pathways to the pathology of SCD should be further investigated.Support or Funding InformationThe authors wish to acknowledge support from NIH grants 1UO1HL117659 and R01HL142604
Molecular Pathogenesis of Bone Degenerative Disease and Associated Inflammatory Processes
Authors: Joshi Priya , Amour Maryanne , Siddiqui Fakiha , Liles Jeffrey , Hoppensteadt Debra , Fareed Jawed , Hopkinson William 
Published: April 2020

IntroductionOsteoarthritis is one of the most common forms of arthritis, which is a result of the degeneration of protective cartilage at the end of bones, resulting in friction in the joints. This is due to a complex pathophysiologic process where inflammation and physical alterations of the bone contribute to the overall pathology. During this process, several mediators of inflammation and matrix degradation are formed. Profiling of some of these mediators and related biomarkers provides a molecular approach to understand the processes involved in the progression of this disease. The aim of this study is to profile various biomarkers of inflammation and hemostatic activation in osteoarthritis patients undergoing total joint replacement.Materials and MethodSeventy‐four EDTA plasma samples were collected from osteoarthritis patients prior to their surgery. The control comprised of a pool prepared from normal male and female EDTA plasma. Such coagulation markers as D‐Dimer, vWF, MP‐TF, PAI‐1, TAFI, and fibronectin were performed using ELISA techniques. Matrix metallopeptidase 9 (MMP‐9) and C‐reactive protein were also measured using ELISA methods. All results were compiled as group means and compared with EDTA controls. Appropriate statistical methods were used to analyze the data.ResultsThe circulating level of D‐Dimer was much higher on average in the patient samples (9178 ± 3.56 ng/ml) in comparison to the control samples (115 ± 85 ng/ml) p<0.05. The concentration of vWF was higher (125 ± 28 %) than in the control sample (104 ± 7.4%) p<0.05. There was not a significant difference between the MP‐TF or TAFI concentrations in the total joint samples vs the controls p<0.05. There was no correlation among any of the tested biomarkers.ConclusionThis data shows that osteoarthritis patients exhibit marked elevation of inflammatory and hemostatic activation biomarkers. Wide variations are noted in the circulating level of these biomarkers in patients. This data suggests that multifactorial processes are involved in the pathogenesis of osteoarthritis. Profiling of inflammatory and thrombotic biomarkers may be helpful in the risk stratification and risk management in these patients.
Numerical method analysis of the activation and time‐dependent inhibition of midazolam metabolism by ticlopidine
Authors: Paragas Erickson Mesina , Nagar Swati , Korzekwa Ken 
Published: April 2020

Drug metabolism plays an important role in the pharmacokinetic and pharmacodynamic properties of drug candidates. In most cases, drug metabolizing enzymes (DMEs) display non‐Michalis‐Menten or atypical kinetics. Atypical kinetics includes activation, autoactivation, partial inhibition, substrate inhibition, and biphasic saturation curves. These kinetics make it difficult to obtain in vitro kinetic constants to be used for in vivo predictions of drug‐drug interactions. Numerical methods offer a unique tool to estimate improved kinetic constants. In this study, we use ordinary differential equations to model the activation and time‐dependent inactivation of midazolam (MDZ) hydroxylation by ticlopidine. Ticlopidine (TCP) is the first thioenopyridine‐based prodrug developed to inhibit platelet aggregation. Time‐dependent inactivation (TDI) assays showed both activation and partial inactivation of midazolam hydroxylation. Co‐incubation of MDZ with different TCP concentrations (0–250 μM) activated the formation of 1‐hydroxymidazolam, increasing kcat by 30%. Additional experiments are currently underway to model the observed activation and time‐dependent inactivation of CYP‐mediated metabolism of MDZ by TCP.Support or Funding InformationFunding: NIGMS grants R01GM114369 and R01GM104178.
Integrative Analysis of Long Non‐Coding RNAs and mRNAs in Lungs of Sickle Cell Mice
Authors: Kang Bum-Yong , Chang Sarah S. , Park Changwon , Archer David R. , Sutliff Roy L. , Hart C. Michael 
Published: April 2020

RationalePulmonary hypertension (PH) is a serious complication of sickle cell disease (SCD) and causes significant morbidity and mortality. Emerging evidence indicates that long non‐coding RNAs (lncRNAs) play a pivotal role in biological processes such as, cellular proliferation, differentiation, and apoptosis. However, the role of lncRNA and mRNA interaction in SCD‐PH pathogenesis has not been defined. We sought to evaluate the expression profiles of lncRNAs and mRNAs in lungs of sickle cell (SS) mice that spontaneously develop PH.Methods and ResultsTotal RNAs were isolated from lungs of SS mice and littermate control (AA) mice at age 15–17 weeks and subjected to lncRNA and mRNA expression profiling using the Arrystar™ lncRNA array (Rockville, MD). Raw signal intensities were normalized in quantile method by GeneSpring GX software (Agilent Technologies, Santa Clara, CA). Volcano plot filtering was used to screen for differentially expressed (DE) lncRNAs and mRNAs with statistical significance (Fold Change >2.0, p< 0.05). To validate DE lncRNAs and mRNAs, 5 lncRNAs and 5 mRNAs were selected for quantitative real‐time PCR (qRT‐PCR). 2302 lncRNAs and 1753 mRNAs were upregulated (Up) and 2546 lncRNAs and 1455 mRNAs were downregulated (Dn) in lungs of SS mice compared to AA mice. Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatics analysis was used for Gene Ontology (GO) enrichment including biological process (BP), molecular function (MF), and cellular component (CC), and pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. GO analysis showed that the altered mRNAs were mainly distinct to negative (Up) or positive (Dn) regulation of biological processes in BP, ion binding (Up) or organic cyclic compound binding (Dn) in MF, and membrane‐bounded organelle and vesicle (Up) and non‐membrane‐bounded organelle and intracellular organelle (Dn) in CC. Pathway analysis revealed that malaria and MAPK signaling pathway were significantly associated with upregulated mRNAs, whereas C‐type lectin receptor signaling pathway, focal adhesion, and JAK‐STAT signaling pathway were related with downregulated mRNAs. LncRNA‐mRNA co‐expression network analysis was evaluated by Coding‐Non‐coding gene Co‐expression network (CNC network) analysis and generated by Cytoscape software (The Cytoscape Consortium, San Diego, CA).ConclusionsThese results identify potential new targets for therapeutic intervention in SCD and provide new insights into SCD pulmonary vascular dysfunction and PH pathogenesis.Support or Funding InformationNIH HL133053 (BYK)
Flavin binding affinity and initial kinetic characterization of DnmZ, a flavin‐dependent N‐oxygenase
Authors: Huynh Katherine , Hardin Taylor , Guzman Lucero , Castillejo Nadia , Palfey Bruce , Vey Jessica 
Published: April 2020

Doxorubicin is an anthracycline chemotherapeutic produced by Streptomyces peucetius. It is an effective chemotherapeutic but has severe side effects and high toxicity, and the details of its biosynthetic pathway are largely unknown. Baumycin is a further decorated form of doxorubicin with an additional seven‐carbon acetal moiety attached to the anthracycline backbone of the molecule. In this work, we report our progress on the characterization of DnmZ, a Class D flavin monooxygenase involved in formation of the baumycin acetal. DnmZ is a N‐oxygenase that oxidizes an sugar‐linked exocyclic amine, triggering a nonenzymatic retro oxime‐aldol cleavage of the hexose. We aim to characterize the kinetics of the DnmZ‐catalyzed reaction between flavin and oxygen using stopped flow spectrophotometry. Results from this research can aid in the development of derivatized anthracyclines and the understanding of the class of enzymes known as the class D flavin monooxygenases.Our transient‐state experiments confirm that DnmZ catalyzes the formation of the C4a‐(hydro)peroxyflavin intermediate (C4a‐OOH). We have identified the appropriate wavelengths to monitor to observe C4a‐OOH formation (378nm) and elimination (450 and 480 nm). With this information we have quantitated flavin binding and determined that DnmZ’s binding affinity for flavin is approximately 13 μM, which compares well with other Class D monooxygenases. We are also investigating the effect of pH (pH 5.5 to 10.5) on C4a‐OOH formation and elimination. Our preliminary results suggest that increasing pH speeds up C4a‐OOH elimination, but has no effect on the intermediate’s formation.Support or Funding InformationCalifornia State University Program for Education & Research in BiotechnologyNIH SCORE 5SC3GM122652
Exploring the Effects of Increased Bacteria Exposure on Late Gestation Fetal Sheep
Authors: Yu Kevin , Keller-Wood Maureen , Wood Charles E. 
Published: April 2020

The concept of the fetal microbiome is beginning to gain traction as new evidence of bacterial presence in fetal tissues arise. With evidence of bacteria in fetal tissues mounting, it becomes important to find out how the fetus responds to increasing presence of bacteria. In our previous studies, we reported that small numbers of bacteria (too few to cause fever or behavioral response) inoculated into the maternal sheep during late gestation can be detected in fetal tissues. In the present study, we hypothesize that transfer of these low numbers of bacteria from mother to fetus stimulate a transcriptomic response in the fetal brain, and that the response is related to inoculation dose. In this study, we used tissues from our previous study in which we inoculated Staphylococcus aureus bacteria into maternal sheep during late gestation. We studied three experimental groups: Naïve (n=6), 300 CFUs (colony forming units injected into the maternal bloodstream) (n=7), and 30,000 CFUs delivered to maternal bloodstream, mouth, and vagina (n=7). Inoculation doses were chosen to ensure low enough dose to not cause fever or anorexia in the sheep. Fetal cerebral cortex samples were collected 3–5 days post inoculation. RNA was extracted and gene expression analyzed using RNAseq. Validation of RNAseq differential expression results was performed using qPCR. Gene expression in the three groups were minimally overlapping in Principal Component Analysis. Using conservative criteria (p<0.05 after Bonferroni correction, we identified 53 differentially expressed genes (DEG) comparing Naïve to 300 CFU and 60 DEG comparing Naïve to 30000 CFU. We conclude that bacteria passing from mother to fetus stimulate transcriptomics responses in the fetal brain. We speculate that this altered gene expression might alter the course of fetal development.Support or Funding InformationThis work was supported by HD033053, AI120195, and HL083810