Project description:The rapid emergence of drug resistant Staphylococcus aureus (S. aureus), in particular, methicillin-resistant S. aureus (MRSA) poses a serious threat to public health globally. Reuse of metal-based antimicrobials stands for one of the most promising strategies to resensitize drug resistant bacteria to conventional antibiotics. Silver (Ag) has been used as an antimicrobial agent since antiquity, yet its molecular mechanism of action is elusive largely owing to technical challenges to identify the direct silver-binding protein targets accountable for its action. Herein, using an in-house developed hyphenated technique LC-GE-ICP-MS, we successfully separated and identified 38 authentic Ag+-binding proteins (Ag+-proteome) in S. aureus at the whole-cell scale for the first time. By integration with bioinformatic analysis and systematic biochemical characterizations, we captured the first snapshot on the dynamic action of Ag+ against S. aureus at the molecular level, i.e., Ag+ primarily targets glycolysis via inhibiting multiple enzymes and induces the elevation of ROS through functional disruption of redox homeostasis system at the late stage, leading to an upregulation of oxidative pentose phosphate pathway (oxPPP) to alleviate Ag+ stress. However, the activation of oxPPP is ultimately futile due to key oxPPP enzymes being inhibited by Ag+. We further validated that Ag+ could inhibit 6-phosphogluconate dehydrogenase, a key target from oxPPP through binding to His185 at the active site and morphing the shape of catalytic pocket by X-ray crystallography. Significantly, the multi-target mode of action of silver is accountable for its suppression of antibiotic selection effects towards S. aureus as well as its sustainable antimicrobial activity. Such a unique mode of action of Ag+ (and silver nanoparticles) led to enhanced efficacy of a broad range of antibiotics and resensitization of MRSA to antibiotics. Our study resolves the long-standing question of the molecular targets of silver in S. aureus and offers a novel insight into the sustainable bacterial susceptibility of silver, providing a potential approach for combating antimicrobial resistance.

Project description:Aims: The sympathetic nervous system regulates numerous critical aspects of mitochondrial function in the heart through activation of adrenergic receptors (ARs) on cardiomyocytes. Chronic β-AR activation causes maladaptive alterations in cardiomyocyte metabolism that contribute to the pathobiology of heart failure. In contrast, mounting evidence suggests that a1-ARs, particularly the a1A subtype, are cardioprotective and may mitigate the deleterious effects of chronic β-AR activation by shared endogenous catecholamine ligands. The mechanisms through which a1A-ARs exert their cardioprotective effects remain unclear. Here we tested the hypothesis that a1A-ARs adaptively regulate cardiomyocyte oxidative metabolism in both the uninjured and failing heart. Methods: We characterized the effects of global α1A-AR genetic deletion on mitochondrial function and metabolism in the uninjured mouse heart using high resolution respirometry, substrate-specific electron transport chain (ETC) enzyme assays, transmission electron microscopy (TEM), proteomics, and lipidomics. We then compared the effects of α1A- and β-AR agonist treatment on ETC enzyme activity and oxidative stress in vivo and in vitro. We subjected wild type and cardiomyocyte-specific α1A-KO mice to permanent left coronary artery (LCA) ligation and used RNAseq to compare the transcriptomic response. Results: We found that isolated cardiac mitochondria from mice with global α1A-AR genetic deletion (α1A-KO) had deficits in fatty acid-dependent respiration and ETC enzyme activity. TEM revealed abnormalities of mitochondrial morphology characteristic of these functional deficits. The selective α1A-AR agonist A61603 (100 ng/kg/d, 3d) enhanced fatty acid oxidation (FAO) in isolated cardiac mitochondria while increasing expression and activity of the mitochondrial trifunctional protein, a critical FAO mediator. The β-AR agonist isoproterenol enhanced oxidative stress in vitro and this adverse effect was mitigated by A61603. RNAseq revealed broad basal deficits in pathways related to mitochondria and OXPHOS in cardiomyocyte-specific α1A-KO mice; these differences were exaggerated by LCA ligation. A61603 enhanced ETC Complex I activity and protected contractile function following myocardial infarction. Conclusions: Collectively, these novel findings position α1A-ARs as critical regulators of cardiomyocyte metabolism in the basal state and suggest that metabolic mechanisms may underlie the protective effects of α1A-AR activation in the failing heart.

Project description:Chang2008 - ERK activation, hallucinogenic drugs mediated signalling through serotonin receptors This model is described in the article: Towards a quantitative representation of the cell signaling mechanisms of hallucinogens: measurement and mathematical modeling of 5-HT1A and 5-HT2A receptor-mediated ERK1/2 activation. Chang CW, Poteet E, Schetz JA, Gümü? ZH, Weinstein H. Neuropharmacology 2009; 56 Suppl 1: 213-225 Abstract: Through a multidisciplinary approach involving experimental and computational studies, we address quantitative aspects of signaling mechanisms triggered in the cell by the receptor targets of hallucinogenic drugs, the serotonin 5-HT2A receptors. To reveal the properties of the signaling pathways, and the way in which responses elicited through these receptors alone and in combination with other serotonin receptors' subtypes (the 5-HT1AR), we developed a detailed mathematical model of receptor-mediated ERK1/2 activation in cells expressing the 5-HT1A and 5-HT2A subtypes individually, and together. In parallel, we measured experimentally the activation of ERK1/2 by the action of selective agonists on these receptors expressed in HEK293 cells. We show here that the 5-HT1AR agonist Xaliproden HCl elicited transient activation of ERK1/2 by phosphorylation, whereas 5-HT2AR activation by TCB-2 led to higher, and more sustained responses. The 5-HT2AR response dominated the MAPK signaling pathway when co-expressed with 5-HT1AR, and diminution of the response by the 5-HT2AR antagonist Ketanserin could not be rescued by the 5-HT1AR agonist. Computational simulations produced qualitative results in good agreement with these experimental data, and parameter optimization made this agreement quantitative. In silico simulation experiments suggest that the deletion of the positive regulators PKC in the 5-HT2AR pathway, or PLA2 in the combined 5-HT1A/2AR model greatly decreased the basal level of active ERK1/2. Deletion of negative regulators of MKP and PP2A in 5-HT1AR and 5-HT2AR models was found to have even stronger effects. Under various parameter sets, simulation results implied that the extent of constitutive activity in a particular tissue and the specific drug efficacy properties may determine the distinct dynamics of the 5-HT receptor-mediated ERK1/2 activation pathways. Thus, the mathematical models are useful exploratory tools in the ongoing efforts to establish a mechanistic understanding and an experimentally testable representation of hallucinogen-specific signaling in the cellular machinery, and can be refined with quantitative, function-related information. This model is hosted on BioModels Database and identified by: MODEL0975191032. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

Project description:Background. The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem / progenitor cells for the other airway cell types. The objective of this study is to better understand basal cell biology by defining the subset of expressed genes that characterize the signature of human airway epithelial basal cells. Methodology / Principal Findings. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium of healthy nonsmokers obtained by bronchial brushings in comparison to the transcriptome of the complete differentiated airway epithelium. This analysis identified the “human airway basal cell signature” as 1,161 unique genes with >5-fold higher expression level in basal cells compared to the differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The human airway basal cell signature displayed extensive overlap with genes expressed in basal cells from other human tissues and murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the airway basal cell signature was characterized by genes encoding extracellular matrix components, and growth factors and growth factor receptors, including genes related to EGFR and VEGFR signaling. However, while human airway basal cells share similarity with basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, integrins, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels. Conclusion / Significance. The human airway epithelial basal cells signature identified in the present study provides novel insights into the ontogeny, molecular phenotype and biology of the stem / progenitor cells of the human airway epithelium. This study was designed to distinguish the transcriptome of the airway epithelium basal cell from that of differentiated airway epithelium. A basal cell signature was derived and analyzed for functional significance. The signature was also evaluated as basal cells differentiated into ciliated epithelium in vitro.

Project description:Background. The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem / progenitor cells for the other airway cell types. The objective of this study is to better understand basal cell biology by defining the subset of expressed genes that characterize the signature of human airway epithelial basal cells. Methodology / Principal Findings. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium of healthy nonsmokers obtained by bronchial brushings in comparison to the transcriptome of the complete differentiated airway epithelium. This analysis identified the “human airway basal cell signature” as 1,161 unique genes with >5-fold higher expression level in basal cells compared to the differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The human airway basal cell signature displayed extensive overlap with genes expressed in basal cells from other human tissues and murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the airway basal cell signature was characterized by genes encoding extracellular matrix components, and growth factors and growth factor receptors, including genes related to EGFR and VEGFR signaling. However, while human airway basal cells share similarity with basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, integrins, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels. Conclusion / Significance. The human airway epithelial basal cells signature identified in the present study provides novel insights into the ontogeny, molecular phenotype and biology of the stem / progenitor cells of the human airway epithelium.

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