bluuebunny/crossref_metadata_2025_split huggingface dataset:
Large and Small Substrate Binding to DesD via ITC Assay Development
Authors: Weaver Madison , Hoffmann Katherine 
Published: April 2020

Desferrioxamines (“dfo”s) are iron‐chelating compounds which have been used medicinally in cases of iron overload. The synthesis of dfoE involves an enzyme called DesD which is a dimer that has an active site on each chain, but the mechanism of it binding both large or small substrates is unknown. We are studying DesD as a model enzyme for the non‐ribosomal peptide synthetase independent siderophore (NIS) pathway and as a potential new drug target. The purpose of this research is to analyze the co‐factor specificity and large substrate verse small substrate specificity of DesD by comparing substrate dissociation constants to analog dissociation constants (KD, where a smaller number correlates to tighter binding). Future drugs will have to out‐compete the substrates in binding, so we will use these studies to determine which substrate components contribute to maximum binding. Techniques in analytical chemistry will be used to regenerate deferoxamines that were bound to cofactors in previous studies with the DesD enzyme. The binding properties of DesD were explored in a 25% glycerol Isothermal Titration Calorimetry (ITC) buffer. Prior experimentation with this protein was conducted in a buffer that resulted in precipitation of DesD out of solution, thus prompting the need for repeated binding assays in a buffer that maintains synthase solubility. GraphPad Prism 8.0 will be used to analyze the KD and occupancy of DesD. High Performance Liquid Chromatography (HPLC) will be used to separate deferoxamines from substrates they were bound to. An Ocean Optics Spectrometer will be used to quantify the deferoxamines by pairing these substrates to a metal compound that will allow us to identify its absorbance and determine if isolation was effective.We confirmed previous results that the KD of DesD binding to ATP is in the M range, compared to non‐ATP cofactors which result in a KD in the M range. Future studies will compare dfoG (substrate) binding to DesD compared to analog dfoB or product dfoE, and also explore smaller substrates, hydroxysuccinylcadaverine (HSC), vs. analogs. Troubleshooting runs have indicated that isolation of the deferoxamines is feasible. It is expected that substrates like dfoG will be separated from ATP and ADP cofactors. We have confirmed that DesD has a strong binding preference for ATP over non‐ATP cofactors in a buffer that maximizes solubility. This data indicates that the phosphates are essential to tight binding in any future competitive inhibitors. Regeneration of each deferoxamine appeared to be a cost‐effective assay that will allow for these compounds to be used in later experimentation. Future work will expand our understanding of critical regions in the binding site, and aid in developing new inhibitors that could block the NIS pathways and be lead drugs for a new class of antibiotics.
Estradiol signaling interacts with Sirt1 in the regulation of autophagy and adipogenesis
Authors: Cheng Zhiyong , Tao Zhipeng , Shi Limin , Zheng Louise 
Published: April 2020

Adiposity is regulated by estradiol signaling and the nutrient sensor Sirt1. It has been recognized that autophagy plays a key role in adipogenesis and metabolism. However, it remains largely unknown whether and how estradiol signaling interacts with Sirt1 in the regulation of autophagy. Here we showed that activation and overexpression of Sirt1 suppressed autophagy in 3T3L1 cells, which was associated with suppressed adipocyte differentiation. Treatment of preadipocytes with estradiol induced Sirt1 and dampened autophagy, thereby suppressing adipogenesis. Consistently, knockdown of Sirt1 induced autophagy and adipocyte differentiation, which was recapitulated by estrogen receptor inhibition or knockout. In addition, knockdown of Sirt1 abolished the effects of estradiol on autophagy and adipogenesis. The modulation of autophagy and adipogenesis by estradiol and Sirt1 was ascribed at least in part to mTOR‐ULK1 cascade. Protein analysis did not show significant changes in the acetylation levels of autophagy regulators (e.g., Atg5, Atg7, or LC3) during Sirt1 modulation. Taken together, our data reveals an estradiol‐Sirt1‐autophagy axis that regulates adipogenesis. Future study will be of interest and significance to determine the potential role of the estradiol‐Sirt1‐autophagy axis in sex‐dependent fat distribution.Support or Funding InformationAmerican Heart Association Grant 18TPA34230082 (Z.C.) and USDA National Institute of Food and Agriculture Hatch Project 1020373 (Z.C.).
Decoding Glycoproteome Remodeling in Sepsis through Integrative Multi‐Omics Analysis and Parts‐Based Data Representation
Authors: Sorrentino James T. , Toledo Alejandro Gomez , Golden Gregory , Diaz-Peña Ramon , Campos Alexandre Rosa , Nizet Victor , Malmstrom Johan , Smith Jeffrey W. , Lewis Nathan E. , Esko Jeffrey D. 
Published: April 2020

Sepsis is a major clinical challenge accounting for unacceptably high‐rates of morbidity and mortality in intensive care units around the world. After many years of extensive research, reliable markers for diagnosis and treatment monitoring remain desperately needed. The plasma glycoproteome offers a suitable readout to monitor systemic alterations in the body. Thus, tracking structural changes in plasma glycoproteins may uncover new molecular windows to target bacterial sepsis. Unfortunately, the analytical challenge posed by the large heterogeneity of plasma carbohydrate and protein components has severely limited advancements in this area. Here we show that Staphylococcus aureus bacteremia induces a dramatic shift of the plasma glycoproteome in a murine model of monobacterial sepsis. Significant changes in the levels of plasma glycoproteins in circulation, as well as their associations with vascular surfaces were revealed through an integrative glycomics, chemical proteomics, and glycoproteomics approach. Additionally, the global and site‐specific glycosylation patterns of the plasma N‐ and O‐linked glycoproteome were significantly altered during sepsis. Deconvolution of these changes through non‐negative matrix factorization methods uncovered the co‐existence of discrete patterns of glycoproteome remodeling associated with specific subgroups of glycoproteins. This included changes in the levels of N‐glycan core‐fucosylation, terminal sialylation, and increased protein deglycosylation, including complete glycan truncations. Furthermore, computational analysis demonstrates that these molecular patterns are sufficient to robustly stratify the samples based on their infection status. In short, we show that multi‐Omics integration has the power to dissect local alterations in the plasma glycoproteome during monobacterial sepsis, possibly reflecting the contribution of discrete biological processes of cellular glycosylation.Support or Funding InformationThis work was supported by grant P01HL131474 to J.D.E. and J.W.S. J.T.S. acknowledges support from the National Institutes of Health, USA (NIH grant T32GM008806) J.W.S. was supported by R01 GM107523. G.G. was supported by Microbial Sciences Graduate Research Fellowship Awards 1‐F17GG and 1‐F18GG.
Polymorphisms in RocA contribute to the molecular pathogenesis of serotype M28 group A streptococcus
Authors: Bernard Paul Edward , Kachroo Priyanka , Eraso Jesus M. , Zhu Luchang , Madry Jessica E. , Duarte Amey , Linson Sarah E. , Saavedra Matthew Ojeda , Cantu Concepcion , Musser James M. , Olsen Randall J. 
Published: April 2020

Two‐component systems (TCSs) are signal transduction proteins that enable bacteria to respond to external stimuli by altering the global transcriptome. Accessory proteins interact with TCSs to fine‐tune their activity. In group A streptococcus (GAS), RocA (regulator of Cov) is an accessory protein that functions with the CovRS (control of virulence regulator/sensor) TCS. Previous data suggest that the N‐terminal predicted transmembrane domains are important for RocA function and interaction with the CovRS TCS. Whole‐genome sequencing analysis of serotype M28 GAS strains collected from invasive infections in humans identified a higher number of missense (amino acid altering) and nonsense (protein truncating) polymorphisms in rocA than expected. We hypothesized that polymorphisms in RocA alter the global transcriptome and virulence of serotype M28 GAS. We used naturally‐occurring clinical isolates with rocA polymorphisms, an isogenic rocA deletion mutant strain, and isogenic rocA polymorphism mutant strains to perform genome‐wide transcript analysis (RNA‐seq), in vitro virulence factor assays, and mouse and nonhuman primate pathogenesis studies to test this hypothesis. Results demonstrated that polymorphisms in rocA result in either a subtle transcriptome change causing a wild‐type‐like virulence phenotype, or a substantial transcriptome change leading to a significantly increased virulence phenotype. Each polymorphism had a unique effect on the global GAS transcriptome. Additionally, in contrast to previously published data on functional domains of RocA, polymorphisms in both the N‐terminal transmembrane domains and the C‐terminal predicted non‐functional histidine kinase ATPase domain result in an altered virulence phenotype suggestive of loss of RocA activity. Taken together, our data show that naturally‐occurring polymorphisms in one gene encoding an accessory protein can significantly alter the global transcriptome and virulence phenotype of GAS, an important human pathogen.Support or Funding InformationFondren Foundation, Houston Methodist Hospital and Research Institute (to JMM and PEB)American Heart Association Predoctoral Fellowship award 19PRE34380820 (to PEB)NIH grants R21AI139369‐01A1 and R21AI146771‐01 (to JMM)
Regulation of Coronary Blood Flow by the Carotid Body Chemoreceptors in Ovine Heart Failure
Authors: PACHEN MRIDULA , Abukar Yonis , Lever Nigel , Ramchandra Rohit 
Published: April 2020

BackgroundThe carotid body (CB) is an arterial chemoreceptor organ located in the carotid bifurcation, which carries out essential functions in cardiorespiratory control. In response to hypoxia, carotid chemosensory discharge produces respiratory, autonomic, and cardiovascular adjustments critical for body homeostasis. Previous studies have shown that chemoreflex sensitivity is augmented animal models of heart failure (HF). However, the role of the chemoreflex in modulating coronary blood flow during HF is not clear. We hypothesized that carotid chemoreceptor stimulation would increase coronary blood flow in both normal sheep and sheep with HF.MethodsHeart failure was induced by the microembolization technique and instrumentation was performed when ejection fraction was below 40%. Both groups of sheep were implanted with a coronary artery flow probe and an arterial pressure catheter. The responses of mean arterial pressure (MAP) and coronary blood flow (CoBF) were determined in conscious sheep following CB stimulation with intracarotid potassium cyanide (KCN; 30μg/kg). To determine the cardiac vagal contribution to the CoBF response, CB activation was repeated following atropine infusion (8 mg bolus followed by 24 mg/h infusion for 30 minutes).ResultsIn control animals, activation of the CB increased MAP (114±1%) and CoBF (116±4%) but coronary vascular conductance (CVC) was unaltered. In the HF group, CB stimulation increased MAP (108±1%), CoBF (126±3%), and CVC (114±2%). The MAP increase was significantly attenuated after atropine infusion in both animal groups, however, the CoBF and CVC responses to KCN were unchanged. These responses persisted when the heart was paced at a constant rate to eliminate the role of altered metabolic demand.DiscussionOur data indicate that in an ovine model of HF, carotid body mediated increases in MAP are attenuated compared to control animals. There was a significant increase in coronary vascular conductance in the HF animals following CB activation which was absent in the normal animals. Overall, our data suggest that the hyperactive carotid body chemoreflex may serve to increase coronary blood flow in the HF condition.Support or Funding InformationHealth Research Council of New Zealand, National Heart Foundation of New Zealand
Generating Phosphorylation Site Antibodies to Study α2A‐Adrenergic Receptor Phosphorylation by G Protein‐coupled Receptor Kinase 2 in Intact Cells
Authors: Barnett Jessica , Hebert Aaron , Weldin Erica , Farina Zachary , Varney Matthew , Guirguis Jeska , Sterne-Marr Rachel 
Published: April 2020

G protein‐coupled receptors (GPCRs) are transmembrane proteins which are activated by the binding of agonist to create a cell signal response. GPCRs are regulated through phosphorylation by G protein‐coupled receptor kinases. We focus on G protein‐coupled receptor kinase 2 (GRK2) and have previously characterized phosphorylation of the β2‐adrenergic receptor (β2‐AR) by this kinase. Currently, we are trying to determine if the same mutations shown to effect β2‐AR phosphorylation are also important for phosphorylation of the α2A‐adrenergic receptor (α2A‐AR). The α2A‐AR plays a role in regulating insulin, neurotransmitter secretion, and is a pharmaceutical target for pain relief. It differs from β2‐AR in that α2A‐AR has a larger third intracellular loop, containing four consecutive serine residues. These residues can be phosphorylated by GRK2 in the presence of agonist, causing a desensitization response. One way to detect receptor phosphorylation is to use antibodies that specifically recognize phosphorylated receptor. Since there is no commercially available antibody that can detect phosphorylation of α2A‐AR, we developed an antibody, RB617, which detects the phosphorylation of two serine residues. Using this antibody, we have developed methodologies to study GRK2 phosphorylation of α2A‐AR in intact COS‐7 cells. We tested the dose‐response of α2A‐AR to epinephrine and determined the EC50 for GRK2‐dependent phosphorylation to be 35 nM. In the future, we plan to assess the effect of GRK2 mutations previously shown to affect β2‐AR phosphorylation to better understand the role of GRK2 in α2A‐AR desensitization.Support or Funding InformationThis work was funded by NSF MCB0744739 and Siena College Center for Undergraduate Research and Creative Activity (CURCA).
Down‐regulation of Circulating miRNAs in Canines with Chronic Kidney Disease may be Indicative of Renal Fibrosis
Authors: Tavener Selena , Panickar Kiran S. , Jewell Dennis E. 
Published: April 2020

Mature microRNAs (miRNAs) are single‐stranded RNAs approximately 22 nucleotides in length. They are generated from short hairpin RNAs that are produced by the ribonucleases, Drosha and Dicer. miRNAs act as post‐transcriptional regulators by base‐pairing with complementary sequences to mRNAs, generally leading to the silencing of mRNA through either degradation or less efficient translation of their target mRNAs. Circulating miRNAs may also serve as biomarkers for renal dysfunction. One characteristic of chronic kidney disease (CKD) is renal fibrosis which results in a progressive loss of renal function, subsequently leading to renal failure. Mechanisms underlying renal fibrosis, generally characterized by excessive extracellular matrix synthesis and deposition, are not clear but deregulation of miRNAs has been intricately linked to renal fibrosis. We assessed the circulating levels of miRNAs using Qiagen’s Canine mScript miRNA PCR array, in the blood of dogs that were clinically diagnosed as having CKD, post‐mortem. Further, end‐of‐life pathology reports indicated interstitial inflammation, fibrosis, and thickening of the Bowman’s capsule. Blood samples were collected at necropsy from dogs with CKD (n=10; 2–17 yr) and control dogs (n=10; 5–13.5 yr). There was a significant decline in the levels of miRNAs cfa‐let‐7a, let‐7c, let‐7f, and let‐7g in dogs with CKD when compared to controls (p<0.05). Down‐regulation of certain let‐7 miRNAs (let‐7a, let‐7b) has been associated with experimental models of induction of renal fibrogenesis. There was also a >2‐fold reduction (NS) in levels of cfa‐miR‐93, cfa‐miR‐122, cfa‐miR‐200a, and cfa‐miR‐204, in CKD when compared to controls. These microRNAs have been demonstrated to have anti‐fibrogenesis effect, and are also down‐regulated in rodent models and in vitro mechanistic models of renal fibrosis leading to increased fibrosis. There was also a down‐regulation of cfa‐miR‐16, which is consistent with its reported role in attenuating kidney injury independent of fibrosis. Taken together with the literature, down‐regulation of these miRNAs may be indicative of a reduction in their role in attenuating renal fibrogenesis and injury. Importantly, the circulating miRNAs may serve as non‐invasive biomarkers for renal fibrosis in CKD and also as nutritional targets for slowing the progression of fibrosis in kidney disease.Support or Funding InformationThis study was funded by Hill’s Pet Nutrition, Inc
Increase in Interleukin‐16, a Pro‐inflammatory Cytokine, and Other Distinct Markers of Lymphocytic Activation, in Kidney Tissue of Cats with Renal Dysfunction
Authors: Panickar Kiran S. , Tavener Selena , Hollar Regina L. , Jewell Dennis E. 
Published: April 2020

Inflammation plays an important role in the initiation and subsequent progression of chronic kidney disease. An increase in inflammatory markers including cytokines as well as markers of oxidative stress is associated with reduced kidney function. Inflammatory markers and the underlying mechanisms that contribute to the pathophysiology of kidney disease are not clear and likely are multifactorial. Another feature associated with renal dysfunction and inflammatory response is the formation of calcium oxalate stones formed from the crystal deposits in the tubular epithelial cells. The aim of the present study was to investigate changes in gene expression in the renal cortex obtained from cats with kidney disease or calcium oxalate stone formers (CaOx) at necropsy in order to identify novel inflammatory biomarkers associated with renal dysfunction. At the time of death the circulating levels of creatinine as well as symmetric dimethyl arginine (SDMA), both markers of kidney decline in cats, were significantly higher in cats with renal disease (n=11) or stone‐forming cats (CaOx, n=12) when compared to controls (n=19). Using RNAseq in kidney tissue, we found a significant increase in the expression of IL‐16, a T‐cell chemoattractant, in both cats with kidney disease (6.72 fold) and stone formers (7.03 fold) compared to controls (both p<0.0001). There was also a significant increase in caspase recruitment domain family member 11 (CARD11), an activator of T and B‐lymphocytes and NFkB, in cats with kidney disease (9.2 fold) and stone formers (10 fold) compared to controls (p<0001). However, some differences between the kidney disease and CaOx groups were also observed. In cats with kidney disease there was an increased expression of LY9 (CD299), found in T and B‐cells and thymocytes, IL2Rg, a marker of lymphocyte activation, and SPP1 (Osteopontin), expressed in activated T and B‐cells (all >6‐fold) when compared to controls (p<0.0001) but such increased expression was not seen in CaOx cats. In summary, while IL‐16 was increased in both groups, our results indicate important molecular differences in the pathogenesis of kidney dysfunction in cats with kidney disease and stone formers, and that distinct lymphocyte markers may be involved in the persistent decline of kidney function. Our studies suggest that the optimal nutritional therapy to slow the progression of kidney dysfunction may be different for cats with kidney disease or stone formers.Support or Funding InformationThis study was funded by Hill’s Pet Nutrition, Inc
Loss of Peptidyl‐Arginine Deiminase 1‐mediated Citrullination Drives Esophageal Epithelial Barrier Impairment in Allergic Inflammation
Authors: Mehta Rishi S. , Rochman Mark , Rothenberg Marc E. 
Published: April 2020

BackgroundCitrullination or deimination is a post‐translational modification in which neutral arginine residues are converted to positive citrulline by members of the peptidyl‐arginine deiminase (PADI) family. PADI1 is an esophageal enriched gene that is significantly downregulated in Eosinophilic Esophagitis (EoE), an eosinophilic‐driven chronic allergic inflammatory disorder characterized by reduced tissue differentiation and impaired esophageal epithelial barrier formation. While the role of PADI1 in deiminating structural proteins has been implicated in epithelial cell populations, including the skin, the functional significance and extent of PADI1‐mediated citrullination in allergic inflammation remains understudied.HypothesisHerein we hypothesize that the loss of PADI1‐mediated citrullination drives esophageal epithelial barrier impairment in allergic inflammation.MethodsPADI1 WT and KO EPC2 immortalized human esophageal epithelial cells generated by CRISPR‐Cas9 were grown in either monolayer or at Air‐Liquid Interface (ALI) to mimic esophageal differentiation. Transepithelial Electrical Resistance (TEER) measurements of EPC2 cells grown at ALI were measured as a function of barrier integrity. Transcriptional, protein and epigenetic analyses were conducted on EPC2 cells grown at ALI treated with or without IL‐13, a T‐cell derived cytokine known to induce responses involved in EoE. Immunofluorescence (IF) was used to visualize structural proteins and assess overall level of citrullination.ResultsPADI1 protein was found to be most highly expressed in the Esophageal Mucosa as compared to all other tissue types, specifically in the suprabasal layer of differentiated esophageal cells. PADI1 RNA expression was decreased eight‐fold (P < 0.0001) in esophageal biopsies of patients with EoE as compared to control patients. Treatment of EPC2 cells with IL‐13 (100 ng/ml) at confluence reduced PADI1 mRNA by three‐fold (P < 0.01). PADI1 KO cells demonstrate altered differentiation as assessed by decreased levels of citrullinated proteins and structural epithelial proteins, including filaggrin, involucrin, desmoglein‐1, E‐cadherin, and cornulin compared with wild‐type control cells.ConclusionOur data suggest that loss of PADI1‐mediated citrullination may drive esophageal epithelial barrier impairment in allergic inflammation.Support or Funding InformationThis work is supported by PHS Grant R01‐AI124355 and the Ohio Physiological Society.