bluuebunny/crossref_metadata_2025_split huggingface dataset:
Untargeted Metabolomics Reveals Abnormal Hepatic Energy Metabolism in β, β‐Carotene‐9′, 10′‐Oxygenase 2 Knockout Mice
Authors: Lyu Yi , Wu Lei , Guo Xin , Davis Abby , Clarke Stephen , Lucas Edralin , Smith Brenda , Wang Weiqun , Medeiros Denis M. , Shen Xinchun , Lin Dingbo 
Published: November 2023

Objectiveβ,β‐carotene‐9′, 10′‐dioxygenase 2 (BCO2) is a mitochondrial protein. Ablation of BCO2 impairs mitochondrial function leading to oxidative stress. The purpose of this study is to define the metabolomic profile and to explore the functional roles of BCO2 in liver energy metabolism.MethodsSix‐week‐old male BCO2 systemic knockout (BCO2−/−, KO), heterozygous (BCO2+/−, Het), and wild‐type (BCO2+/+, WT) mice in the 129S6 background fed a chow diet were used in this present study. Untargeted liver metabolomics analysis was conducted using ultrahigh performance liquid chromatography‐tandem mass spectroscopy (UPLC‐MS/MS) and gas chromatography‐mass spectroscopy (GC‐MS). Western blot was employed to confirm the altered metabolic pathways.ResultsThe results of the metabolomics profiling and bioinformatics analysis revealed that there were distinct differences in metabolites in liver of KO mice, compared to WT and Het mice. However, no marked difference was found in the Het liver, compared to WT. Oxidation of amino acids, such as leucine, tryptophan, and lysine, which is dependent on mitochondria, was restricted in KO mice. On the other hand, metabolites associated with the TCA cycle, including succinate, fumarate, and malate, were significantly increased in KO liver. Moreover, metabolites related to carnitine biosynthesis, which is important for lipid metabolism, were significantly decreased in KO mice. Biomarkers of oxidative stress, such as 4‐hydroxynonenal‐glutathione and S‐lactoylglutathione, were elevated in KO mice compared to WT and Het. Additionally, stable arachidonate (AA) derivatives of the lipoxygenase (lox) pathway were elevated in liver, suggesting that AA‐mediated inflammatory processes were stimulated in BCO2 KO mice. The results also showed that sterol metabolism and the conversion of cholesterol to primary and secondary bile acids were altered in BCO2 KO mice.ConclusionsBCO2 is essential to macronutrient metabolism in the liver of mice. The ablation of BCO2 causes alteration of energy metabolism, resulting in oxidative stress and inflammation in the liver of mice.
Activity transcription factor 3 suppressed LPS‐induced adipocyte inflammation in experimental sepsis
Authors: Wang Yu‐Hsin , Cheng Ching‐Feng , Lai Pei‐Fang 
Published: November 2023

Endotoxemia, a state of excessive endotoxin (lipopolysaccharide, LPS) in the circulation, frequently results in fatal sepsis. Activating transcription factor (ATF)‐3 is a member of the ATF/cAMP‐response element‐binding protein family of transcriptional factors. It is also induced by stress condition in a variety of tissues, including the adipose tissue. The aim of this study is to assess the possible protective role of ATF3 in adipose tissue inflammation of LPS‐induced polymorphonuclear leukocyte recruitment in experimental sepsis. ATF3‐knockout (KO) and wild‐type mice (WT) were injected with LPS (5 mg/kg, ip) to induce endotoxemia. Multifaceted approaches including the in vivo animal endotoxemic mice model for examining biochemical analysis, the expression of inflammatory mediators, and pathophysiological examinmation in the adipocytes were investigated. Results demonstrated that LPS enhanced ATF3 protein expression in WT control mice, but not in ATF3‐KO group in the adipocytes. We found that the protein expression of iNOS and the numbers of polymorphonuclear leukocyte were significantly increased in the adipose tissue of KO mice, but not observed in WT mice after LPS induction. Furthermore, restored ATF3 gene expression by transfection of adeno‐associated virus (AAV) in the KO mice, the increased WBC levels, iNOS expression, and LPS‐mediated polymorphonuclear leukocyte recruitment were significantliy suppressed that comparing to the WT mice. These results suggested that ATF3 gene may play a critical role to overcome the adipose tissue inflammation in experimental sepsis.Support or Funding Informationsupported by MOST and Tzu Chi Medical center
Splenic Natural Killer Cell Phenotypes Are Heritable and Correlate with Lifespan
Authors: Bumgardner Sara A , Zhou Yang , Jiang Zixuan , Coe Erica J , Pazdro Robert 
Published: November 2023

Natural Killer (NK) cells are cytotoxic innate immune cells integral to the host defense against viruses and neoplastic cells. With age, NK cells undergo immunosenescence, yielding functional impairments in immune surveillance. While numerous mechanisms have been explored to explain NK cell dysfunction with age, it is currently unknown if the aged NK cell phenotype is heritable. In this study, we evaluated conventional splenic NK cell maturation phenotypes, as defined by CD27 and CD11b surface expression, from 15 classical inbred and 4 wild‐derived aged murine strains. We found that both total and mature NK cell proportions, along with their median surface density of CD27 and CD11b, are highly heritable across strains. We additionally identified total splenic NK cell proportions as an independent predictor of median lifespan in mice. When select strains were compared to younger controls, NK cell phenotypes were variably stable across age, with C57BL/10J and A/J strains demonstrating phenotypic evidence of NK cell immunosenescence. These results reveal strong narrow‐sense heritability of murine NK cell phenotypes and intriguing models for future mechanistic and functional NK cell studies.Support or Funding InformationResearch was funded by the University of Georgia Office for the Vice President for Research and the College of Family and Consumer Sciences.
Silica‐Triggered Multi‐Organ Autoimmune Gene Expression in Lupus‐Prone Mice is Ablated by Docosahexaenoic Acid Consumption
Authors: Bates Melissa A. , Gilley Kristen N. , Jackson‐Humbles Daven N. , Harkema Jack R. , Holian Andrij , Pestka James J. 
Published: November 2023

While heredity is a primary predisposing factor for autoimmunity, the exposome can critically influence latency and severity of autoimmune diseases such as systemic lupus erythematosus (SLE). We have previously demonstrated that airway exposure to the toxicant crystalline silica (cSiO2) triggers early loss of self‐tolerance in the lung thereby accelerating onset of systemic autoimmunity and exacerbates glomerulonephritis in the female NZBWF1 mouse, a widely used SLE model. Remarkably, dietary supplementation with the omega‐3 polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA), abrogates cSiO2‐induced lung inflammation, systemic autoimmunity and nephritis in this model. We hypothesized here that cSiO2‐triggered inflammation in the lung precedes systemic autoimmunity and nephritis in NZBWF1 mice and is a primary target for the prophylactic action of DHA. Beginning at 6 wk of age, female NZBWF1 mice were fed an isocaloric AIN‐93G diet containing 0.0, 0.4, or 1.0 % DHA. Two wk later, mice were intranasally instilled with 1.0 mg cSiO2 once per week for 4 wk and maintained on experimental diets until sacrifice. At 13, 16, 19 and 22 wk of age, cohorts were sacrificed and lung, spleen, and kidney were collected. Total RNA from these tissues for each cohort was isolated and purified, and gene expression levels were determined with the nCounter® PanCancer Immune Profiling for mouse, a multiplexed method for immune profiling of 800 target genes. cSiO2‐induced elevations in gene expression were first evident in the lung in cohorts from 13 wk and later whereas in spleen and kidney elevations were seen at 16 wk and thereafter. Genes observed to upregulated by cSiO2 included chemotactic factors, cytokines, B cell receptors, and remarkably, genes involved in interferon regulation. Importantly, cSiO2‐triggered expression of these genes was dose‐dependently suppressed by DHA in all three tissues over time. Taken together, marked attenuation cSiO2‐induced elevations of inflammatory and immune genes by DHA in the lung, spleen and kidney suggests that supplementation with this omega‐3 PUFA might be used to mitigate environmental triggering of autoimmune disease.Support or Funding InformationLupus Foundation of America, Inc.
Pathological Role of Two Chemokines RANTES and MIF in Ischemic Stroke
Authors: Liu Yu‐Chuan , Tang Sung‐Chun , Liou Houng‐Chi , Tu Huang‐Ju , Kang Kai‐Hsiang , Liou Horng‐Huei , Jeng Jiann‐Shing , Fu Wen‐Mei 
Published: November 2023

WHO reported that around 15 million people have a stroke worldwide every year. Among them, ischemic stroke accounts for about 80 percent in all cases, but the treatment of ischemic stroke is still limited to tPA injection. Therefore, developing the new strategy to treat stroke is important and urgent. Cytokines and chemokines were found to contribute to the infarct size in stroke. This study aimed to find out the pathological roles of two chemokines, Macrophage migration inhibitory factor (MIF) and Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES) in stroke.In our study, the human blood samples from stroke patients before and after tPA injection were evaluated by human cytokine array and ELISA kits. Wistar rats were subjected to transient middle cerebral artery occlusion (tMCAo) and treated with MIF or RANTES via femoral vein injection right after tMCAo. Twenty‐four hours after tMCAo, the infarct volume was evaluated by TTC staining and the neurological function was determined by the modified set of the mNSS. In addition, the blood‐brain barrier (BBB) of tMCAo rat was evaluated by Evans blue dye. Primary neuron and adult rat brain endothelial cell (ARBEC) were also subjected to oxygen‐glucose deprivation (OGD)/reoxygenation to examine the cell viability or BBB integrity after treatment of MIF or RANTES.In patients, the concentrations of MIF and RANTES were significantly increased following ischemic stroke before tPA injection. In Wistar rats, the exogenous administration of MIF (3.3 μg/kg) after tMCAo significantly increased the infarct volume; however, the RANTES‐treated (10 μg/kg) rats decreased infarct size after ischemia/reperfusion. To investigate how MIF and RANTES influence the outcome after ischemic stroke, we examined the BBB permeability after tMCAo. It was found that MIF increased the BBB permeability; however, RANTES reduced the BBB leakage after ischemia/reperfusion. In ARBEC culture also showed that MIF increased, but RANTES reduced the BBB leakage after OGD/reoxygenation.In conclusion, the levels of MIF and RANTES were elevated after ischemia. MIF increased the neuron damage in tMCAo rats; in contrast, RANTES exerts a neuroprotective effect after tMCAo. Our study indicates that MIF and RANTES may be a good drug target for developing drugs for the treatment of ischemic stroke.Support or Funding InformationThis work was supported by the Ministry of Science and Technology grants, 104‐2321‐B‐002‐033, Taiwan.
Divergent Influence of Binge Drinking and Subsequent Intermittent Stress on Drinking Behaviors and Select Brain Protein Levels in Male and Female Mice
Authors: Devaud Leslie L , Garcia Andres , Perius Megan , Finn Deborah A 
Published: November 2023

Alcohol misuse and abuse are serious problems in our society and stress may increase the risk for escalation of drinking from recreational to problematic. The present study explored the effect of prior binge drinking on subsequent drinking behaviors following intermittent predator odor stress exposure, which is a model of traumatic stress. Adult male and female C57BL/6 mice had either 7 binge ethanol sessions (binge EtOH) or drank water (water group). Following a 30‐day abstinence period, all mice were provided free access to 10% ethanol (10E) and water via lickometers for 4 weeks, with 30 min exposure to predator odor during the middle 2 weeks (2X/week). At 24 hrs after the final drinking session, mice were euthanized, and brains were harvested and frozen for later Western Blot analyses vs separate naïve controls. Dissected prefrontal cortex (PFC) and hippocampus tissues were assayed for levels of CRF‐R1 (corticotropin releasing factor receptor 1), P450scc (an enzyme involved in steroidogenesis) and the GABAA receptor (GABAAR) alpha1 subunit. Whereas intermittent predator odor significantly increased anxiety‐like behaviors in male and female mice, measured on the elevated plus maze, it significantly increased 10E licks over baseline to a greater extent (78%) in the binge EtOH males versus the water males (27%). In contrast, females had no change in drinking following stress, regardless of prior binge drinking experience or not. Western blot analyses showed divergent responses across proteins and sex. In PFC, there were no significant differences between the binge EtOH and water groups. CRF‐R1 levels were unchanged across male or female groups, but there was a > 25% decrease in P450scc levels in males and a 20% increase in females, vs naïve controls. Levels for the GABAAR alpha1 subunit did not change in males but decreased by 20% in stressed females vs naïve. In contrast, male hippocampal tissue showed different responses between the stressed binge EtOH and water groups, vs naïve. CRF‐R1 levels were increased by 25% in water males with no change in binge EtOH animals. P450scc and GABAAR alpha1 subunit levels were unchanged in water groups but decreased by 13% and increased by 22% in the binge EtOH groups, respectively. Taken together, these data reinforce the importance of assessing sex differences as the synergistic effect of binge drinking and stress on alcohol intake occurred only in male mice. Additionally, the divergent neurochemical responses in males and females suggest that stress exerted the greatest effect in the PFC while binge EtOH exerted the greatest effect in the hippocampus.Support or Funding InformationSupported by BX001070 and BX002966 (DF) from the Department of Veteran Affairs
Structure‐Guided Design for Agonist Selectivity with α7‐nAChRs
Authors: Camacho‐Hernandez Gisela Andrea , Kaczanowska Katarzyna , Harel Michal , Beck Christina , Doan Lisa , Binding Hanna , Taylor Palmer 
Published: November 2023

Nicotinic acetylcholine receptors (nAChRs) are ligand‐gated ion channels present in the peripheral and central nervous systems. Pre‐ and post‐synaptic nAChRs modulate CNS functions, and ligands selective for nAChR subtype may form useful therapies for disorders associated with development and aging of the nervous system. We have developed a small sub‐group of 4,6 di‐substituted 2‐aminopyrimidines that interact with the acetylcholine binding protein (AChBP) and activate α7‐nAChRs. These agents show Kd and Ka values for both AChBP binding and α7‐nAChR activation between 10 and 70nM. To examine the structural basis of agonist selectivity, we employ measurements of ligand binding to AChBP, and analysis of ligand binding poses from X‐ray crystal structures, along with α7‐nAChR activation on intact cells in culture. Crystal structures suggest a distinct binding pose is required for α7‐nAChR activation enabling us to define the molecular determinants giving rise to activation and selectivity for the α7 subtype. By combining the picolyl substitution at the 4‐position of the pyrimidine and departing from the symmetry of the molecule, we are able to discriminate between α7 activation and generalized binding at the subunit interface. The 4,6‐disubstituted 2‐aminopyrimidines have high selectivity for α7‐nAChR. Crystallography studies using the soluble acetylcholine binding protein (AChBP), a surrogate protein of the extracellular domain of α7‐nAChR, reveal three distinctive binding interactions and poses of nicotinic agonists: 1) cation‐π interactions for quaternary amines and an aromatic nest of side chains at the subunit interface, 2) stabilization of secondary amines and imines by a hydrogen bond from its protonated basic nitrogen to the backbone carbonyl of a conserved tryptophan on the principal subunit face, 3) interaction of less basic multi‐ring pyrimidine‐pyridine heterocycles at the binding interface. This new family of 2‐aminopyrimidines enables us to define receptor subtype selectivity and achieve a structural landscape for α7 selective activation amongst a plethora of CNS nAChR subtypes.Support or Funding InformationNIH Grant GM 18360‐44
Identifying Receptor‐Ligand Interactions That Drive Gβγ‐Dependent Signaling
Authors: Cameron Robert Bruce , Beeson Craig Bruce , Schnellmann Rick 
Published: November 2023

Understanding differences in ligand bias enable more targeted application of pharmacotherapy to various disease states. Our laboratory has previously demonstrated that in renal proximal tubule cells (RPTC), the beta‐2 adrenergic receptor agonist formoterol induces mitochondrial biogenesis, while the beta‐2 adrenergic receptor agonist clenbuterol does not. We hypothesized that formoterol and clenbuterol engage distinct signaling programs by engaging in different interaction profiles with the beta‐2 adrenergic receptor. We examined Gαs‐ and Gβγ‐dependent signaling in RPTC following stimulation with 30 nM formoterol and 30 nM clenbuterol and found that both ligands caused significant accumulation of cAMP but that only formoterol caused a significant increase in Akt phosphorylation. To understand the structural basis of these differences in signaling, formoterol and clenbuterol were docked to the orthosteric binding site of both inactive (3NYA, 3NY8, 5D5B) and active (4DLE, 4LDL, 4LDO) crystal structures of the beta‐2 adrenergic receptor. For each pose, receptor‐ligand interactions were tabulated at each structural feature. Each interaction was assigned a value of +1 for formoterol and −1 for clenbuterol, and interactions were summed for all six crystal structures. The methoxyphenyl group of formoterol showed a greater likelihood of aromatic interactions with W109 and V114 and contact interactions with F193. Interestingly, the formamide and hydroxyl groups of formoterol exhibited a greater interaction frequency with the backbone of C191, but the amino group of clenbuterol was more likely to engage in strong interactions at that site. Similarly, the secondary amino groups of both formoterol and clenbuterol interacted with D113, but formoterol was more likely to do so with strong hydrogen bond or ionic interactions. Overall, the longer and larger functional groups of formoterol allowed it to stretch across the binding pocket of the beta‐2 adrenergic receptor to simultaneously interact with TM3, TM5, and ECL2 in a manner distinct from clenbuterol and may drive the differences in signaling between formoterol and clenbuterol. These interaction profiles provide key structural data for the development of beta‐2 adrenergic receptor agonists that selectively activate specific signaling programs.Support or Funding InformationR.B.C. is supported by F30 DK104550 and T32 GM008716 (National Institutes of Health). C.C.B. is supported by P20 GM103542 (National Institutes of Health). R.G.S. is supported by R01 GM084147 (National Institutes of Health) and 1BX000851 (Department of Veterans Affairs).
Identification of Binding Sites for Small Molecule Modulators of Gβγ Signaling
Authors: Fisher Isaac James , Connelly Sara , Dumont Mark , Smrcka Alan 
Published: November 2023

G‐protein coupled receptors (GPCRs) transduce extracellular stimuli to intracellular signaling events via G‐proteins and β‐arrestins and are targeted by up to 40% of FDA approved drugs. Targeting proteins downstream of the GPCR, such as G‐protein β, allows for selective modulation of specific pathways downstream of GPCRs. Inhibition of interactions between Gβγ and specific effectors has shown to have therapeutic potential in diseases such as heart failure and inflammation (Lehmann et al., 2008. and Casey et al., 2010.)Previously, our laboratory identified small molecules that bind to a common site for protein‐protein interactions on Gβ. These compounds selectively modulate Gβγ‐dependent activity of various downstream effectors. We hypothesize that these compounds differentially modulate Gβγ‐mediated signals due to binding to specific subsets of amino acids on Gβ. To find the binding site for these compounds, we used a random mutagenesis screen in yeast. The yeast mating response to α‐factor is mediated by the Gβγ homolog, Ste4p. This pathway when activated can inhibit yeast growth making it an attractive tool for examining the inhibition of βγ and homologs using small molecules.Gallein, a well‐characterized Gβγ inhibitor, inhibited the α‐factor/Ste4P mediated mating response pathway and it suppressed α‐factor induced growth arrest. To screen for residues on Ste4p that bind gallein, we set up a yeast strain where α‐factor stimulates growth on media lacking histidine and showed that gallein inhibits growth of this strain in the presence of α‐factor. We randomly mutagenized ste4p and 500,000 variants were screened for colonies that grew in the presence of gallein. Two classes of mutations were found. Class I mutants had high basal activity suggesting that the mutations decreased the affinity of Ste4P for Gpa1p. Indeed, these mutations were located at positions known to be important for mammalian Gα binding to Gβγ. Class II mutants did not exhibit high basal activity. To test whether these mutants were resistant to gallein inhibition because they lack a gallein binding site, we examined the concentration dependence for gallein inhibition in a reporter assay. The IC50's for the Class I mutants were not different from wild‐type suggesting that resistance was not conferred by a loss of gallein binding. We are in the course of examining class II mutants. One double mutant, G182R, C282E, showed an increased IC50 for gallein while other remain to be examined. All of these mutations map to a region on a critical Gβγ protein‐protein interaction surface.Finding gallein binding specific residues will allow us to define an inhibitor binding site on Gβ, and may allow for rational design of novel therapeutic inhibitors. In addition, it will help us understand the mechanism for selective inhibition of signaling downstream of Gβγ by small molecules.