bluuebunny/crossref_metadata_2025_split huggingface dataset:
NLRP3 Inflammasome Products IL‐1beta and IL‐18 Have A Direct Effect on Cardiomyocytes
Authors: Enni Jacqueline Bella Anderson , Ninh Van , Miyamoto Shigeki , Brown Joan Heller 
Published: April 2020

ObjectiveWe previously demonstrated a significant role for NLRP3 inflammasome activation in cardiomyocytes in cardiac inflammation, remodeling and contractile dysfunction in response to stressors such as Angiotensin II infusion and pressure overload. There is limited data regarding responses of cardiomyocytes to interleukin‐1 beta (IL‐1beta) and interleukin 18 (IL‐18), which are generated as a result of NLRP3 activation. The goal of this study was to determine the effects of IL‐1beta and IL‐18 on cardiomyocytes.MethodsIsolated Neonatal Rat Ventricular Myocytes (NRVMs) were treated for various times with a range of doses of recombinant IL‐1beta or IL‐18. Western Blotting was used to measure the activation state of protein kinases and downstream signaling pathways. Quantitative Polymerase Chain Reaction (q‐PCR) was used to assess the expression of pro‐inflammatory cytokines and chemokines in cardiomyocytes.ResultsWestern blotting showed a significant increase in phosphorylation of multiple protein kinases and molecules that regulate apoptosis and other inflammatory functions after IL‐1beta and IL‐18 treatment, with maximal responses seen at 1ng/ml IL‐1beta and 10ng/ml IL‐18. Phosphorylated mitogen‐activated protein kinases (MAPKs) including JNK and P38 as well as p‐STAT3 were increased several fold at 10 and 30 mins of treatment with either cytokine. q‐PCR analysis of NRVM mRNA revealed increases in gene expression of multiple pro‐inflammatory cytokines and chemokines in cells treated with either IL‐1beta and IL‐18. CXCL‐10, TGF‐beta, IL‐18, and NLRP3 were all upregulated by both IL‐1beta or IL‐18. Interestingly, upregulation of CCL2, IL‐6, CXCL1, and TNF‐alpha resulted from treatment with IL‐1beta but not with IL‐18. We are currently analyzing the effects of IL‐1beta and IL‐18 on cell viability to further determine the direct effect of these products of the NLRP3 inflammasome in regulating cardiomyocyte apoptosis basally or in response to stressors.ConclusionsWe have previously shown that the cardiomyocyte is a source of proinflammatory cytokines and chemokines, including IL‐1beta and IL‐18. Our results suggest the possibility that IL‐1beta and IL‐18 activate a pro‐inflammatory positive feedback loop in cardiomyocytes which enhances and sustains cardiac inflammation, contributing to cardiac remodeling under stress conditions.
R‐(‐)‐2,5‐dimethoxy‐4‐iodoamphetamine (DOI)‐induced Vocalizations and Observable Behavior in Nonhuman Primates
Authors: Wakeford Alison G.P. , Kohut Stephen J. 
Published: April 2020

Many psychiatric disorders include elements of dysfunctional social behavior. Understanding the pharmacological underpinnings of social behavior in a nonhuman primate species that demonstrates a repertoire of complex behavior (e.g., aggression or affiliative behavior) may provide insights into novel medication approaches aimed at restoring social deficits. Specifically, squirrel monkeys (Samiri boliviensis) emit a rich array of vocalizations that provide information on socio‐emotional and behavioral states. These vocalizations often co‐occur with readily observable behaviors that are easily quantifiable, permitting analyses of various aspects of social behavior. Previous studies have indicated that affiliative, or prosocial, vocalizations (pulsed calls and chucks) and behavior (huddling) may be modulated by serotonin 2A (5HT2A) receptors. To begin to understand how 5HT2A receptor activation influences social behavior, we examined the effects of R‐(‐)‐2,5‐dimethoxy‐4‐iodoamphetamine (DOI), a potent and moderately selective 5HT2A agonist, on affiliative vocalizations and observable behavior in nonhuman primates. Pair‐housed squirrel monkeys (2 males, 2 females) were transferred to an observation chamber outside of the colony and vocalizations and huddling were recorded during test sessions. Saline or DOI (0.01 or 0.06 mg/kg, IM) was administered prior to 30 min sessions. Results show that vocalizations, in particular pulsed calls, were increased following administration of 0.01 mg/kg DOI (438 ± 28) relative to saline (190.5 ± 21.5); a higher dose of 0.06 mg/kg DOI elicited a number of vocalizations that were not different from saline (201.5 ± 88.5). Frequency of huddling was unaffected by saline or DOI administration, however, increases in general activity were observed following DOI administration at both doses in comparison to saline. A preliminary examination of potential sex differences suggests that there were no differences between males and females in huddling or total number of vocalizations, though males produced more alarm calls at the highest dose of DOI tested. These data suggest that 5HT2A receptor activation is sufficient for increasing affiliative vocalizations, but may not change other indices of prosocial behavior, such as huddling. Future studies will expand the list of behaviors captured to include positional analyses of each subject, as well as evaluating the contribution of other 5HT receptors to specific dimensions of prosocial behavior.Support or Funding InformationResearch support provided by R01DA048150 (SJK).
High‐yield production of hematopoietic growth factors by tobacco BY‐2 cells for ex vivo production of red blood cells
Authors: Xu Jianfeng , Wang Xiaoting , Wright Tristen 
Published: April 2020

Ex vivo generation of clinically available red blood cells (RBCs) from hematopoietic stem cells (HSCs) represents a promising approach for overcoming the limitations associated with the use of donor’s blood, such as shortage of blood and risk of transfusion‐transmitted infections. Both expansion and differentiation of stem cells are highly reliant on growth and differentiation factors, with the activity and purity of these molecules being critical to successful research and clinical applications. The overarching goal of this project is to develop a novel plant cell‐based bioproduction platform for high‐quality (i.e. animal‐free and endotoxin‐free) erythropoietic growth factors (eGFs) that facilitate mass production of RBCs from HSCs at significantly reduced cost. In addressing the low productivity bottleneck of plant cell culture technology, this proposal aims to leverage a unique, plant‐specific, O‐glycosylation process called “hydroxyproline (Hyp)‐O‐glycosylation” for de novo design and engineering of
Hyp‐O‐
glycosylated
peptides (HypGPs”) that presumably function as a molecular carrier in boosting secretion of conjoined eGFs from cultured plant cells. This will dramatically increase the secreted yields of plant cell‐derived eGFs and simplify the product purification process. Three key eGFs essential for HSCs expansion and differentiation: erythropoietin (EPO), stem cell factor (SCF) and interleukin 3 (IL‐3), are expressed in tobacco BY‐2 cells as fusion with a strategically designed HypGP tag. The secreted protein yields, molecular structure, and Hyp‐O‐glycosylation of the HypGP‐tagged eGFs are characterized. The bioactivities of individual HypGP‐tagged eGF in stimulating proliferation and differentiation of relevant hematopoietic cell lines, includingTF1 erythroleukemic cell and umbilical cord blood‐derived CD34+ cell, are evaluated to establish a suitable design of HypGP tag (size and orientation) for each eGF. A scalable cell culture system fortified sorely with plant cell‐produced eGFs is developed for production of RBCs from CD34+ HSC cells. This project provides a proof‐of‐concept for ex vivo manufacturing of stem cell‐derived RBCs with plant cell‐produced eGFs.Support or Funding InformationNational Science Foundation Directorate for Engineering [1605564]; National Institutes of Health‐Arkansas INBRE [P20GM103429]
Kisspeptin Amplifies GLP‐1 Potentiation of Insulin Secretion by a cAMP Independent Pathway
Authors: Lynch Ronald M. , Aragaki Kai A. , Harnois Emily , Vagner Josef , Weber Craig 
Published: April 2020

Glucagon Like Peptide‐1 (GLP‐1) and Kisspeptin (KP) receptors are co‐expressed on pancreatic b‐cells, and activation of both receptors potentiates glucose stimulated insulin secretion (GSIS). Unlike GLP‐1, KP has a low affinity for receptor activation (EC50 GLP‐1 ~20 nM vs KP >1 uM). The effects of GLP‐1 and a 10 amino acid analog of KP (KP10) on insulin secretion and cell signaling were studied in the INS 832/3 cell line. By itself KP10 has no effect on insulin secretion in the absence of glucose, but doubled GSIS (GSIS = 37.7 +/− 6.7; +KP10 = 69.2 +/− 1.9 ng/ml/80min) with an EC50 of ~5–10 mM. At saturating doses, KP10 (100 mm) and GLP‐1 (100 nM) had similar effects on GSIS, and in combination had an additive effect (GLP‐1 = 49.0 +/− 2.0, GLP‐1 + KP10 = 63.5 +/− 5.6 ng/ml/80min) consistent with activation of independent downstream signaling pathways. Neither GLP‐1 or KP‐10 altered intracellular Ca2+ levels at basal or activating glucose concentrations. GLP‐1 increased cAMP production (165.0 +/− 17.4 % over control), whereas KP‐10 was without effect. Conversely, pretreatment with KP‐10 enhanced glucose stimulated oxygen consumption and mitochondrial NADH fluorescence, although both responses were inconsistent. Therefore, the effects of KP on b‐cell metabolic activity needs to be investigated in more detail.We also developed a bivalent ligand composed of GLP‐17‐36 and KP10 binding domains, linked with a 3‐mer of proline/glycine repeats, to evaluate if linking KP10 to a high affinity ligand could promote KP10 activity at low concentrations by presumably increasing its local membrane concentration after GLP‐1 binding. The dimer also did not activate secretion in the absence of glucose, but doubled GSIS much like the individual monomers with an EC50 ~200nM; that is, an additive response was not obvious. Currently, we are determining the ability of GLP‐1/KP10 to initiate downstream signaling from both ligands to resolve the question: is KP active in the GLP‐1/KP at concentrations where monomeric KP10 is not.Support or Funding InformationFunded by the American Diabetes Association and Juvenile Diabetes Research Foundation
Endothelial sodium channel (EnNaC) activation contributes to mineralocorticoid receptormediated increases in coronary artery and cardiac fibrosis/stiffness leading to diastolic dysfunction in obesity
Authors: Aroor Annayya Rao , DeMarco Vincent , Bender Shawn , Jia Guanghong , Habibi Javad , Yang Yan , Manrique-Acevedo Camila , Lastra Guido , Whaley-Connell Adam , Jaisser Frederic , Sowers James , Hill Michael 
Published: April 2020

BackgroundWhile obesity, induced by an industrialized or Western diet (WD), has been shown to impact structural and functional properties of both coronary arteries and myocardium, the exact mechanisms by which these interact to cause diastolic dysfunction (DD) are uncertain. We hypothesized that the mineralocorticoid receptor (MR) activation in coronary vascular endothelial cells (ECs) impairs endothelial function via EC Na+ channel, EnNaC, activation. Further, we posited that such coronary dysfunction initiates increased myocyte stiffness and fibrosis ultimately leading to DD. The objective was to demonstrate that WD‐induced DD is dependent on EnNaC activation.MethodsTo cause MR activation, aldosterone was administered to female or male mice (C57BL6) by osmotic minipump for 3 wks. Further, female wild‐type and EC MR deficient mice (2–3 mths of age) or low aldosterone implanted as subcutaneous pellet were fed a WD, high in saturated fat and fructose (16 wks). In an additional experiment, female wild‐type and EnNaC α subunit deficient mice (3–4 mths of age) were fed the WD (12–16 wks). Vascular function was assessed in isolated coronary arteries by pressure myography, cardiac and perivascular fibrosis was evaluated by picosirius red (PSR) staining and diastolic function was assessed by echocardiography. Using in vitro cellular approaches, intrinsic cardiomyocyte stiffness was measured by atomic force microscopy (AFM) and EnNaC activity (Na+ current) determined by whole cell patch clamp.ResultsAldosterone infusion caused impaired endothelium‐dependent relaxation and coronary vascular fibrosis, which were prevented by the MR antagonist, spironolactone (1 mg/kg/day). WD feeding was associated with DD, but not systolic dysfunction, that was prevented by either spironolactone or EC‐specific MR deletion. Furthermore, DD was accompanied by increased vascular EnNaC currents and increased stiffness of both ECs and cardiomyocytes, which were attenuated by EC MR deletion. Finally, WD‐induced increases in activation of coronary EnNaC, EC stiffness and cardiomyocyte stiffness, together with DD, were suppressed in EnNaC α deficient mice.ConclusionsCollectively, these results demonstrate that MR activation (aldosterone infusion and diet‐induced) elicits coronary vascular dysfunction associated with tissue and cellular cardiac changes and DD. Further, activation of EnNaC, a known target of MR signaling, promotes coronary EC‐dependent cardiomyocyte stiffness, cardiac fibrosis and impairs DD. Therapeutic targeting to prevent EnNaC activation may be a viable approach for the management of obesity‐associated diastolic dysfunction.
Uncovering Sex‐specific Roles of Farnesoid X Receptor in Heme Metabolism and Liver Proliferation
Authors: Dean Angela Elizabeth , Anakk Sayeepriyadarshini 
Published: April 2020

Nuclear receptor, Farnesoid X Receptor (FXR), regulates bile acid homeostasis and has been shown to promote liver regenerative response. Serendipitously, we uncovered a distinct role for FXR in mediating liver injury response in female compared to male mice challenged with 3,5‐diethoxycarboncyl‐1,4‐dihydrocollidine (DDC, a heme biosynthesis disruptor). As expected, the DDC diet led to liver injury as measured by elevated serum ALT and AST levels in both control and FxrKO mice. Furthermore, accumulation of bile plugs and ductular reaction was visible in the WT mice, whereas both of these histological hallmarks were dramatically reduced in FxrKO mice. Despite similar histology, the proliferative response in the hepatocytes were different between both the sexes of FxrKO animals. Loss of Fxr resulted in increased hepatocyte size and low‐grade Ki‐67 positive hepatocyte nuclei even under basal conditions. But upon DDC challenge, FxrKO female livers displayed a dramatic induction of Ki‐67 positive staining even though no bile duct injury was visible. This finding correlated well with increased expression of cyclins. On the contrary, Ki‐67 staining was comparable between male FxrKO livers fed either chow or DDC diet, which matches with lower injury. We are actively investigating the crosstalk of estrogen signaling with FXR in controlling liver proliferation. At the molecular level, genes regulating the cell cycle (Ccnd1, Ccna, Ccnb1) were induced upon injury in the male livers in an Fxr‐dependent manner, whereas this response was not observed in female livers. Expression of Cyp2b10 and Cyp3a11, which are known detoxification genes, were induced with DDC treatment. To test if the absence of bile plugs in FxrKO mice is secondary to reduced heme synthesis in these livers, we examined the heme biosynthetic pathway. We mined previously published data and found strong Fxr occupancy and binding to 6 out of the 8 genes involved in the heme synthesis pathway including Alas1, which is the rate‐limiting step in heme biosynthesis. In summary, FXR may regulate heme metabolism and modulate hepatocyte proliferation in a sex‐specific manner.Support or Funding InformationResearch Scholar Grant TBE 132315
A single residue exerts two contrasting effects on autoinhibition of HCN channels
Authors: Page Dana A. , Li Jessica , Jung Matthew , Ela-Alim Allaa , Young Edgar C. 
Published: April 2020

Hyperpolarization‐activated cation (HCN) channels regulate electrical activity in the brain and heart in a cAMP‐dependent manner. The voltage‐gating of these channels is mediated by a transmembrane (TM) region but is additionally regulated by direct binding of cAMP to a cyclic nucleotide‐binding (CNB) fold in the cytoplasmic C‐terminal region. In the unliganded form, the CNB fold mediates an autoinhibitory mechanism that kinetically and thermodynamically stabilizes the closed state, and cAMP binding relieves this inhibition. This autoinhibition is an important regulatory mechanism, relief of which provides a molecular mechanism for how cAMP potentiation occurs. The magnitude of this autoinhibition can be calculated by comparing thermodynamic or kinetic parameters of a channel to the parameters from a channel that is autoinhibition‐free due to truncation of the CNB fold. Recently, I have found that the magnitude of autoinhibition is regulated by interdomain interactions between the C‐terminal region and TM region structures. Specifically, the magnitude of both kinetic and thermodynamic autoinhibition was augmented by replacing the TM region of the HCN2 channel with the TM region of HCN4. I hypothesized that interdomain interactions between the HCN4 TM region and charged residues in the C‐linker, which connects the CNB fold to the TM region, can modify the magnitude of autoinhibition. I used site directed mutagenesis to identify that the E457 residue formed an electrostatic interaction with the HCN4 TM region to stabilize the open state. This residue also has an important role in both the kinetic and thermodynamic components of the autoinhibition mechanism. A charge reversal mutation (E457R) limited the thermodynamic effect of autoinhibition while augmenting the kinetic effect of autoinhibition. Overall, this work suggests that the molecular mechanisms of autoinhibition must include participation of TM region structures and provides functional evidence for interdomain interactions that facilitate HCN channel regulation by CNB fold‐mediated mechanisms.Support or Funding InformationNSERC, Simon Fraser University
Calcium‐Sensing Receptor (CaSR) Modulates Hyperoxia‐Induced Airway Hyperreactivity In a Neonatal Mouse Model of Bronchopulmonary Dysplasia
Authors: Sopi Ramadan B. , Mayer Catherine A. , Martin Richard J. , Pabelick Christina B. , Prakash Y. S. , MacFarlane Peter M. 
Published: April 2020

Supplemental O2 is a primary mode of respiratory therapy for preterm infants. Numerous studies, however, have shown that supplemental O2 may contribute to the pathophysiology of airway hyperreactivity (AHR) associated with wheezing disorders such as asthma later in childhood. The calcium‐sensitive receptor (CaSR) is a potent modulator of smooth muscle contractility and may be an underlying feature of childhood asthma. In the present study, we investigated whether CaSR is involved in neonatal hyperoxia induced AHR in a mouse model of BPD. We hypothesize that inhibition (CaSR antagonism), downregulation of expression (siRNA), or deletion (CaSR KO mice) of CaSR will attenuate the AHR induced by hyperoxia in neonatal mice. Newborn C57BL/6 wild‐type (background) or CaSR−/− (knockout) mice were randomized on the second day of life and assigned to room air (21% O2) or hyperoxic (40% O2) groups and exposed for seven days, then recovered in room air for two additional weeks. At day P21, AHR was assessed in vitro using precision‐cut living lung slice preparations in response to increasing doses of methacholine (MCh, 0.25–8 μM). Wild‐type lung slices were pre‐incubated for 48 h in a CaSR siRNA sequence (40 nM) or scrambled RNA as a negative control. In another set of experiments, slices were pre‐incubated in a CaSR antagonist – NPS 2143 (10 nM) for 1 h. Airways were imaged and airway reactivity was expressed as percent change from baseline airway lumen area (± SEM). Neonatal hyperoxia significantly increased airway contractile responses to MCh in wild‐type mice with a mean maximal effect (Emax) of 68.4±8.7% decrease in airway lumen area compared to room air controls (47.3±5.8%, p<0.001), indicating AHR. Pre‐incubation (48hrs) with siRNA targeting CaSR or pharmacologic inhibition (NPS 2143, 1hr incubation) of CaSR reversed the hyperoxia‐induced AHR and the contractile responses were normalized to control level, with hyperoxic groups Emax of 44.0±5.2% and 41.3±3.1%, respectively. AHR was not observed in CaSR knockout mice suggesting resistance to hyperoxia exposure. These data suggest that CaSR plays an important role in the long‐term effects of neonatal hyperoxia on AHR. We speculate that CaSR may be a novel therapeutic target to reverse airway hyperreactivity in former preterm infants who had received prior supplemental O2 therapy.Support or Funding InformationFunding: NIH: HL R01 138402 and NIH R01 HL 056470
Exploring the Autoinhibitory Domain (AID) of the Electrogenic Na/HCO3 Transporter NBCe1‐B, and Binding Motifs for the Cytosolic Protein IRBIT
Authors: Lee Seong-Ki , Boron Walter F. 
Published: April 2020

The electrogenic Na/HCO3 cotransporter NBCe1 is expressed widely throughout the body, and plays crucial roles in trans‐epithelial HCO3− transport (e.g., renal proximal tubule, PT) and regulation of intracellular pH (e.g., cells of central nervous system, CNS). The five known NBCe1 variants are: NBCe1‐A variant, mainly in PT; B, ubiquitous; C, CNS; and D and E, murine reproductive organs. For each variant, the extreme NH2‐terminus (Nt) markedly affects transport activity. For the A and D variants, the initial 41 amino acids (aa) are unique and, at least in A, confer high transport activity. For the B, C, and E variants, the initial 85 aa are likewise unique and, at least in B and C, confer low transport activity. Among these variants, at least B interacts with IRBIT [inositol trisphosphate (IP3)‐receptor binding protein released with IP3], which binds to the unique Nt and markedly enhances transport activity. In a previous study, we examined NBCe1‐B (e1B) residues 28–62, inclusive, and found that the cationic cluster (residues 40–48, inclusive) is an essential element of the AID that confers low activity to e1B. The purpose of the present study is to: (a) determine whether other portions of the unique Nt of e1B (i.e., residues 2–27 and 63–85, inclusive) contribute to the AID, (b) better characterize the cationic cluster, and (c) refine the structural requirements of the IRBIT‐e1B interaction under physiological conditions. Our approach is to co‐express in Xenopus oocytes one of several e1B constructs ± super‐IRBIT (sIRBIT, which lacks the PP1 binding site), followed by two‐electrode voltage‐clamping, extracellular surface biotinylation, and NeutrAvidin pull‐down of plasma‐membrane proteins. As assayed by western blot, we find that sIRBIT co‐purifies with biotinylated e1B protein, but only for e1B constructs with an intact AID (i.e., low‐activity constructs that sIRBIT strongly stimulates). For example, sIRBIT does not copurify with the Δ940‐48 e1B construct, which lacks the cationic cluster (i.e., AID disrupted). We find that residues 63–85, inclusive, are unnecessary for both the AID and for sIRBIT binding. Currently, we are using alanine scanning to explore residues 2–27, inclusive. When we replace the normal cation cluster—mostly made of arginine (R) residues—with increasing numbers (N) of lysine residues (KN) or histidine residues (HN), we observe that (a) the KN and HN constructs also produce autoinhibition, (b) IRBIT relieves the autoinhibition, and (c) sIRBIT co‐purifies with the KN and HN constructs, and (d) the amount of co‐purified sIRBIT gradually increases with increasing N for both KN and HN constructs, as is the case with RN constructs. Thus, the cationic cluster—whether made up of RN, KN, or HN—is essential both for autoinhibition and sIRBIT binding.Support or Funding InformationThe Office of Naval Research, N00014‐15‐1‐2060,N00014‐16‐1‐2535